JP2023550721A - Additives to reduce non-specific interactions between fluorescent polymer conjugates and cells in biological samples - Google Patents

Abstract

The present disclosure relates to methods and compositions for reducing or eliminating nonspecific binding of at least one dye conjugate to cells in a biological sample. The dye conjugate is contacted with at least one zwitterionic or anionic surfactant prior to, during, or after contacting the dye conjugate with the blood sample to improve the biological properties of the dye conjugate. results in a substantial reduction in non-specific binding to cells in the target sample.

Description

This application was filed as a PCT International Patent Application on November 12, 2021, and claims priority benefit to U.S. Provisional Application No. 63/113,703, filed on November 13, 2020. , which application is hereby incorporated by reference in its entirety.

Background Polymer dye conjugates offer excellent performance that can be utilized in bright, monochromatic or multicolor flow cytometry assays. Generally, polymer dye conjugates exhibit high brightness due to their unique and complex structure. However, that same unique and complex structure may also introduce some significant limitations. This disclosure addresses these limitations.

Summary By their nature, polymer dye conjugates are capable of binding non-specifically to cells in biological samples, such as monocytes and granulocytes in peripheral blood samples. Non-specific binding may lead to misinterpretation and result in false positive inferences. For example, if a polymer dye conjugate comes into contact with blood during the analysis of cell markers, the conjugate may bind non-specifically to cells, such as monocytes and/or granulocytes, thereby falsely identifying them as a positive population. It may result in signals that can be interpreted or may single out populations other than the desired ones.

The present disclosure provides solutions to these and other problems associated with the use of polymer dye conjugates. In some embodiments, the present disclosure provides a composition for reducing or eliminating nonspecific binding of a dye conjugate to cells in a biological sample, comprising: a dye conjugate as described herein; Compositions including a conjugate and a surfactant are provided.

In some embodiments, the present disclosure provides a method for reducing or eliminating nonspecific binding of at least one dye conjugate to cells in a biological sample, the method comprising: contacting at least one dye conjugate and at least one zwitterionic surfactant before, during, or after contacting with the target sample, the contacting comprising: providing a reduction in non-specific binding of at least one dye conjugate of the method. In some embodiments, the present disclosure provides a method for reducing or eliminating nonspecific binding of at least one to cells in a blood sample, the method comprising: prior to contacting a dye conjugate with the blood sample; contacting at least one dye conjugate and at least one anionic surfactant during or after contacting, the contacting comprising: contacting at least one dye conjugate in the blood sample; A method is provided that includes the step of providing a reduction in non-specific binding. The compositions and methods of the present disclosure reduce or eliminate non-specific binding of polymeric or non-polymeric dye conjugates to monocytes and/or granulocytes in a blood sample.

A method for reducing or eliminating nonspecific binding of at least one dye conjugate in a biological sample, such as a blood sample, the method comprising: prior to contacting the polymer dye conjugate with the biological sample, the method comprises: contacting at least one dye conjugate with at least one zwitterionic or anionic surfactant during or after contacting, the contacting comprising at least one zwitterionic or anionic surfactant in the biological sample. A method is provided that includes reducing non-specific binding of one polymeric dye conjugate.

In some embodiments, the biological sample may be a blood sample. In some embodiments, the cells may be leukocytes and the reduction in non-specific binding may include reduction in non-specific binding to leukocytes in the blood sample. In some embodiments, the leukocytes are selected from the group consisting of monocytes and granulocytes.

In some embodiments, the method includes adding a surfactant to the polymeric dye conjugate prior to contacting the polymeric dye conjugate with a biological sample, such as a peripheral blood sample.

In some embodiments, the method includes adding a surfactant to the blood sample prior to contacting with the polymer dye conjugate.

Surfactants have the formula:
R ^1' [CO-X(CH ₂ ) _j ] _g - [N ⁺ (R ^2' ) (R ^3' )] _k - (CH ₂ ) _f - [CH(OH)CH ₂ ] _h -Y ^- ( During the ceremony,
R ^1′ is saturated or unsaturated C _5-24 alkyl;
X is NH, NR ⁴ ′, where R ^4′ is C _1-4 alkyl, O, or S;
j is an integer from 1 to 10;
g is 0 or 1;
R ^2' and R ^3' are independently C _1-4 alkyl;
k is 0 or 1;
Hydroxyl is optionally substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl;
f is an integer from 0 to 4;
h is 0 or 1;
Y is COO, SO ₃ , OPO(OR ^5' )O, or P(O)(OR ^5' )O, where R ^5' is H or C _1-4 alkyl)
When k=0, the surfactant may be in acidic form or its sodium or potassium salt.

In some embodiments, the surfactant has the formula:
R ^1' [CO-X(CH ₂ ) _j ] _g -N ⁺ (R ^2' )(R ^3' )-(CH ₂ ) _f -[CH(OH)CH ₂ ] _h -Y ^- ,
(In the formula,
R ^1′ is saturated or unsaturated C _5-24 alkyl;
X is NH or NR ^4' , where R ^4' is C _1-4 alkyl, O, or S;
j is an integer from 1 to 10;
g is 0 or 1;
R ^2' and R ^3' are independently C _1-4 alkyl;
Hydroxyl is optionally substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl;
f is an integer from 1 to 4;
h is 0 or 1;
Y is COO, SO ₃ , OPO(OR ^5' )O or P(O)(OR ^5' )O, where R ^5' is H or a C _1-4 alkyl residue)
zwitterionic surfactant compounds.

Zwitterionic surfactants have the formula:
R ^1' -N ⁺ (CH ₃ ) ₂ -CH ₂ COO ^- ;
R ^1' -CO-NH(CH ₂ ) ₃ -N ⁺ (CH ₃ ) ₂ -CH ₂ COO ^- ;
R ^1' -N ⁺ (CH ₃ ) ₂ -CH ₂ CH(OH)CH ₂ SO ₃ ^- ; or R ^1' -CO-NH-(CH ₂ ) ₃ -N ⁺ (CH ₃ ) ₂ -CH ₂ CH (OH)CH ₂ SO ₃ ^-
It may be a compound of

In some embodiments, the zwitterionic surfactant is almond amidopropyl betaine, apricot amidopropyl betaine, avocado amidopropyl betaine, babasamidopropyl betaine, behenamidopropyl betaine, behenyl betaine, canolamide propyl ( canolamidopropyl betaine, capryl/capramidopropyl betaine, carnitine, cetyl betaine, cocamidoethy betaine, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, cocobetaine, cocohydroxy Sultaine, coco/oleamidopropyl betaine, coco sultaine, decyl betaine, dihydroxyethyl oleyl glycinate, dihydroxyethyl soy glycinate, dihydroxyethylstearyl glycinate, dihydroxyethyl tallow glycinate, dimethicone propyl PG-betaine, drucamidopropyl hydroxysultaine, hydrogenated tallow betaine, isostearamidopropyl betaine, lauramidopropyl betaine, lauryl betaine, lauryl hydroxysultaine, lauryl sultaine, milk amidopropyl Betaine, milkamidopropyl betaine, myristamidopropyl betaine, myristyl betaine, oleamidopropyl betaine, oleamidopropyl hydroxysultaine, oleyl betaine, oliveamidopropyl betaine, palmamidopropyl betaine, palmitamidopropyl betaine, palmitoyl Carnitine, palm kernel amidopropyl betaine, polytetrafluoroethylene acetoxypropyl betaine, ricinolamide propyl betaine, sesamidopropyl betaine, soyamidopropyl betaine, stearamidopropyl betaine, stearyl betaine, tallow amidopropyl betaine, It may be selected from the group consisting of tallow amidopropyl hydroxysultaine, tallow betaine, tallow dihydroxyethyl betaine, undecylenamidopropyl betaine, and wheat germ amidopropyl betaine. In some embodiments, the surfactant is lauryl betaine.

In some embodiments, the surfactant has the formula:
R ^1' [CO-X(CH ₂ ) _j ] _g -(CH ₂ ) _f -[CH(OH)CH ₂ ] _h -Y ^- (in the formula,
R ^1′ is saturated or unsaturated C _5-24 alkyl;
X is NH, NR ⁴ ′, where R ^4′ is C _1-4 alkyl, O, or S;
j is an integer from 1 to 10;
g is 0 or 1;
R ^2' and R ^3' are independently C _1-4 alkyl;
Hydroxyl is optionally substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl;
f is an integer from 0 to 4;
h is 0 or 1;
Y is COO, SO ₃ , OPO(OR ^5' )O or P(O)(OR ^5' )O, where R ^5' is H or C _1-4 alkyl); It may be a surfactant compound, and the anionic surfactant may be in acidic form, or in the form of its sodium or potassium salts. In some embodiments, f=0. In some embodiments, f=1. In some embodiments, f=3. In some embodiments, f=4. In some embodiments, Y is COO or _SO3 . In some embodiments, R ^2' and R ^3' are methyl.

Anionic surfactants have the formula R ^1' -CO-N( _CH3 ) _-CH2 - ^COO- ; or ^R1' -CO-N( _CH3 ) _-CH2 - _SO3- ;
R ^1' may be saturated or unsaturated C _5-24 alkyl) and their sodium or potassium salts. In some embodiments, R ^1' can be saturated or unsaturated C _7-19 alkyl, or C _11-17 alkyl.

In some embodiments, the anionic surfactant is N-lauroylsarcosine, sodium lauroylsarcosinate, sodium palmitoylsarcosinate, sodium stearoylsarcosinate, sodium N-methyl-N-(1-oxotetradecyl)-glycine. salt, sodium caproyl sarcosinate, sodium capryloyl sarcosinate, N-methyl-N-(1-oxo-9-octadecen-1-yl)-glycine, sodium salt, sodium oleoyl sarcosinate, and linoleoyl sarcosine It may be selected from the group consisting of sodium chloride. In some embodiments, the anionic surfactant is N-lauroylsarcosine.

In some embodiments, the polymeric dye conjugate has formula III:
(In the formula,
each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers;
each optional M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap modifying monomers, optionally substituted ethylene, and ethynylene;
Each optional L is a linker part;
each G ¹ and G ² is independently selected from an unmodified polymer terminus and a modified polymer terminus;
a, c, and d define the mol% of each unit, each of which may be repeated uniformly or randomly, each a being a mol% from 10 to 100%, and each c being a mol% of 10 to 100%. , 0 to 90% mol%, and each d is 0 to 25% mol%;
each b is independently 0 or 1;
each m is an integer from 1 to about 10,000)
The binding partner is conjugated to a polymeric dye having the structure:

In some embodiments, A includes a DHP moiety. In some embodiments, A includes a fluorene moiety. In some embodiments, A includes DHP and a fluorene moiety.

In some embodiments, the polymeric dye conjugate has Formula I:
(In the formula,
each X is independently C or Si;
each Y is independently CR ¹ R ² or SiR ¹ R ² ;
Each R ¹ is independently an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a water-solubilizing moiety:
And;
Each R ² is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, Ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, or water-solubilizing moiety
And;
each R ³ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups;
each Z is independently selected from the group consisting of C, O, and N;
each Q is independently selected from the group consisting of a bond, NH, ^NR4 , and _CH2 ;
Each subscript n is independently an integer from 0 to 20;
Each M is a unit capable of modifying the polymer bandgap, uniformly or randomly distributed along the polymer backbone; L is a linker; G ¹ and G ² are hydrogen, halogen , alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxy, azides, sulfonates, phosphates, boronic acid-substituted aryls, boronic acids Ester substituted aryl, boronic ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene; amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N - one or more pendant chains terminating with a functional group selected from hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and their protecting groups conjugated to the binding partner each independently selected from the group consisting of aryl or heteroaryl substituted with . In some embodiments, L is uniformly or randomly distributed along the polymer backbone, including amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl , hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and their protecting groups conjugated to the binding partner with one or more pendant chains terminating with a functional group selected from the group consisting of is an aryl or heteroaryl group; a, c, and d each define the mol% of each unit in the structure, which may be repeated uniformly or randomly, and a is 10 c is 0 to 90% mol%; each d is 0 to 25% mol%; each b is independently 0 or 1; m is an integer from 1 to about 10,000; each n is independently an integer from 1 to 20.

A binding partner can be a molecule or a molecular complex capable of specifically binding a target analyte. A binding partner may be a protein, affinity ligand, antibody, or antibody fragment. In some embodiments, the binding partner is a monoclonal antibody, a polyclonal antibody, an immunoglobulin, an immunologically active portion of an immunoglobulin, a single chain antibody, a Fab fragment, a Fab' fragment, and an F(ab') fragment, and scFv fragments.

Compositions are provided that include a polymeric dye conjugate; an aqueous buffer; and a zwitterionic or anionic surfactant. The composition may include zwitterionic or anionic surfactants at concentrations below the critical micelle concentration (CMC). In some embodiments, the surfactant is 0.05 to 0.25% (w/v), 0.06 to 0.20% (w/v), or 0.08 to 0.16% (w/v). The concentration may be (w/v). The aqueous buffer may contain additional additives selected from the group consisting of protein stabilizers, preservatives, and additional surfactants. The composition may exhibit reduced non-specific binding of the polymer dye conjugate to leukocytes in the sample after exposure to a blood sample and flow cytometry analysis. The reduction in non-specific binding may be compared to the same composition without zwitterionic or anionic surfactants. The leukocytes may be selected from the group consisting of monocytes and granulocytes.

FIG. 1A shows a plot of fluorescence intensity as a function of wavelength for fluorene (FF), dihydrophenanthrene (DD), and fluorene-DHP (DF) polymer dyes.

FIG. 1B shows a graph of the absorption spectra of fluorene (Fl-Fl) and dihydrophenanthrene (DHP-DHP) polymers. The DHP-DHP polymer (black curve) shows a lambda max (λmax) at 390 and 410 nm, while the Fl-Fl (gray curve) polymer shows a lambda max (λmax) around 400 nm.

Figure 2 shows unstained blood cells (upper panel); blood cells stained with a polymeric dye and without surfactant (lower left panel); and blood stained with a composition containing a polymeric dye and a surfactant. Flow cytometry point plot of cells (bottom right) is shown. Blood samples were stained using the antibody-free fluorescent polymer dye SN v605 and analyzed on a flow cytometer. The surfactant-free polymeric dye showed non-specific binding to monocytes/granulocytes (bottom left). Polymeric dyes containing EMPIGEN BB® showed substantial reduction in non-specific binding to monocytes/granulocytes (bottom right).

Figure 3 shows blood cells without polymer dye conjugate (top panel) and blood cells stained with SN 605-CD20 conjugate and with detergent (bottom left panel), and SN 605-CD20 conjugate. A dot plot is shown for blood cells stained with detergent and without detergent (bottom right panel). With the use of detergent, the percentage of non-specifically bound granulocytes decreased (see the "P2" gate in the dot plot). Also, the functional aspects of the conjugate did not change as the percentage of positive population was similar in both cases (see "P1" gate in the dot plot).

Figure 4 shows the median fluorescence intensity of monocytes in the presence and absence of detergent for two lots of polymer dye conjugate (SN v605-CD20) compared to unstained monocytes (autofluorescence). Figure 3 shows a bar graph of MdFI values. In the presence of detergent, non-specific interactions on monocytes were substantially reduced for both Lot 1 and Lot 2 SN605 CD20 conjugates.

Figure 5 is a bar graph of MdFI values of granulocytes in the presence and absence of surfactant for two lots of polymer dye conjugate (SN v605-CD20) compared to unstained granulocytes (autofluorescence). shows. In the presence of detergent, non-specific interactions towards granulocytes were reduced for both Lot 1 and Lot 2 SN605 CD20 conjugates.

Figure 6 shows blood cells without polymer dye conjugate (top left) and blood cells stained with SN v786-CD103 conjugate and Empigen BB® surfactant (bottom left) and SN v786-CD103 conjugate. A dot plot of gated, surfactant-free blood cells (top right) is shown. The dot plot compares one of the claimed polymers to BV786-CD103, a tandem fluorescent dye (bottom right) (available from Becton Dickinson). In the presence of detergent, the percentage of non-specifically bound granulocytes and monocytes decreased (see "P1" and "P2" gates, respectively, in the dot plot).

Figure 7 is a bar graph of MdFI values of monocytes in the presence and absence of detergent for two lots of polymer dye conjugate (SN v786-CD103) compared to unstained monocytes (autofluorescence). shows. In the presence of surfactant, non-specific binding of the polymer conjugate to monocytes was substantially reduced for both lots of polymer dye conjugate.

Figure 8 shows a bar graph of MdFI values of granulocytes in the presence and absence of surfactant for two lots of polymer conjugate compared to unstained granulocytes (autofluorescence). In the presence of surfactant, non-specific binding of the polymer conjugate to granulocytes was substantially reduced for both lots of polymer dye conjugate.

Figure 9 shows point plots of blood cells stained with SN v605-CD20 conjugate with detergent and SN v605-CD20 conjugate without detergent (top panel), bottom left panel. The non-ionic surfactant Tween®-20 and the bottom right panel is the non-ionic surfactant Pluronic F-68. The percentage of non-specifically bound monocytes was not reduced using the nonionic surfactants Tween®-20 and Pluronic F-68 (“Non-specific monocytes” in the dot plot). Please check the gate).

Figure 10 shows blood cells without dye conjugate (top left panel); blood cells stained with SN v605-CD20 conjugate and with BSA (top right panel); stained with SN v605-CD20 conjugate and oxidized BSA. Point plots of blood cells containing BSA-Cy5-ox (bottom left panel) and stained with SN v605-CD20 conjugate (bottom right panel) are shown. The percentage of non-specifically bound monocytes and granulocytes was not substantially reduced using the protein blocker (see "P1" gate in the dot plot).

Figure 11 shows unstained and stained samples for SN v428 CD19 (Figure 11, upper panel), SN v428 CD22 (Figure 11, lower panel), and SN v428 CD25 (Figure 11, middle panel) specificity, respectively. Figure 3 shows three graphs showing the effect of surfactant concentration on negative monocytes (MFI) of donor 1 (D1) and donor 2 (D2) blood samples. The SN conjugate showed lower non-specific monocyte interactions in the presence of 0.06 to 0.20% Empigen BB® than in the absence of detergent.

Figure 11 (continued) shows unstained and stained data for SN v428 CD19 (Figure 11, top panel), SN v428 CD22 (Figure 11, bottom panel), and SN v428 CD25 (Figure 11, middle panel), respectively. Three graphs are shown showing the effect of surfactant concentration on negative monocytes (MFI) of sample donor 1 (D1) and donor 2 (D2) blood samples; Shown as % negative monocyte MFI. Samples stained with BD polymer dye conjugate have a lower percentage of nonspecific monocyte interactions in the presence of 0.06 to 0.20% Empigen BB® than in the absence of detergent. It was shown that

Figure 12 (9 graphs) shows negative granulocytes (top three panels), positive lymphocytes (middle panel), and % positive lymphocytes without Empigen BB® sample (bottom three panels). Figure 2 shows the effect of surfactant concentration on (panel). Unstained and stained blood samples from donor 1 (D1) and donor 2 (D2) are shown. Negative effects on granulocytes in the presence of 0.06 to 0.20% surfactant compared to the absence of surfactant for CD19 BD, CD25 BD, and CD22 BD polymer dye conjugates, respectively. A slightly lower interaction was shown (top three panels). For the CD19 BD, CD25BD, and CD22 BD polymer dye conjugates, respectively, the positive lymphocyte data were similar or slightly higher in the presence of surfactant compared to the absence of surfactant. (middle three and bottom three panels).

Figure 13 shows donor 1 (top panel) and donor 2 (bottom panel) blood samples of detergent-free, 0.06%, 0.12%, and 0.2% Empigen BB®. SS/FL9 staining pattern for SN v428 CD19 lot number D19-094 polymer dye conjugate at 0.5 μg/test and CD19 BV-421 conjugate (Becton Dickinson) at its commercial dose with surfactant. Shows a point plot.

FIG. 14 shows donor 1 (top panel) and donor 2 (bottom panel) blood samples without surfactant, with 0.06%, 0.12%, and 0.2% surfactant. Figure 3 shows a dot plot of the SS/FL9 staining pattern for SN v428 CD25 lot number D19-107 polymer dye conjugate at 0.5 μg/test and CD25 BV-421 conjugate (Becton Dickinson) at its commercial dose.

FIG. 15 shows donor 1 (top panel) and donor 2 (bottom panel) blood samples without surfactant, with 0.06%, 0.12%, and 0.2% surfactant. Point plots are shown for the SN v428 CD22 lot number D19-109 polymer dye conjugate at 0.5 μg/test and the CD22 BV-421 conjugate (Becton Dickinson) at its commercial dose.

Figure 16 shows a dot plot containing the percentage of dead cells up to 0.2% detergent. CD19-SNv428D19-094 without EMPIGEN BB® (negative control) and with 0.06%, 0.12%, and 0.2% EMPIGEN BB® were stained with 7-ADD. Whole blood samples from Donor 1 and Donor 4 were tested to assess the percentage of dead cells in each condition. Whole blood samples stored for more than 24 hours were included as a positive control for 7-AAD staining (left panel, 12% dead cells). The percentage of dead cells was not substantially increased by the presence of up to 0.2% EMPIGEN BB® when compared to samples without detergent.

Figure 17A shows a dot plot of a single color conjugate-free peripheral blood sample, and it is clear that no population is observed in the CD20+ gate.

FIG. 17B shows the presence of CD20-SN v605 monochromatic conjugate in a buffer composition containing BSA, sodium azide, Pluronic™ F-68 (PF-68), and Empigen BB® as additives. Positive control point plots of peripheral blood samples are shown below. When compared to the negative control point plot (Figure 17C), the population % in the gates for "monocyte non-specific binding" and "granulocyte non-specific binding" were significantly reduced, respectively, and Figure 3 shows the effectiveness of Empigen BB® in eliminating or reducing non-specific binding.

FIG. 17C shows a negative control point plot of a peripheral blood sample in the presence of CD20-SN v605 monochromatic conjugate in a buffer composition containing only BSA, PF-68, and sodium azide as additives.

FIG. 17D shows peripheral blood in the presence of CD20-SN v605 monochromatic conjugate in a buffer composition containing BSA, sodium azide, PF-68, and NLS (0.16% w/v) as additives. A test point plot of the sample is shown.

FIG. 17E shows peripheral blood in the presence of CD20-SN v605 monochromatic conjugate in a buffer composition containing BSA, sodium azide, PF-68, and NLS (0.08% w/v) as additives. A test point plot of the sample is shown.

DETAILED DESCRIPTION OF THE DISCLOSURE Reference will now be made in detail to certain specific embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings and examples. While the disclosed subject matter will be described in conjunction with the recited claims, it is understood that the illustrated subject matter is not intended to limit the scope of the claims to the disclosed subject matter. Will.
General disclosure

The present disclosure generally includes at least one surfactant and at least one polymeric dye conjugated to a binding partner (e.g., an antibody), e.g., a fluorescent polymeric dye conjugated to the binding partner. The present invention relates to compositions and methods for detecting analytes in samples using the compositions. More specifically, the present disclosure provides a method for reducing or eliminating nonspecific binding of at least one polymeric dye conjugate in a biological sample, such as a blood sample, the method comprising: contacting the at least one polymeric dye conjugate and at least one zwitterionic or anionic surfactant before, during, or after contacting the biological sample, e.g., a blood sample. wherein the contacting results in reduced non-specific binding of at least one polymeric dye conjugate to cells, such as leukocytes in a blood sample. Surfactants may be added to the blood sample prior to contacting. Surfactants may be added to the polymer dye conjugate prior to contacting with the biological sample.
definition

Abbreviations used herein have their conventional meanings within the chemical and biological arts.

The singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. .

The term "and/or" refers to and includes any and all possible combinations of one or more of the associated listed items. Unless otherwise specified, the phrase "room temperature" refers to 18 to 27°C.

Unless otherwise specified, the term "percent" or "%" refers to percent by weight.

All patents, patent applications, and publications mentioned herein are incorporated by reference in their entirety.

The term "analyte" refers to a molecule, compound, or other constituent in a sample. Analytes can include, but are not limited to, peptides, proteins, polynucleotides, organic molecules, sugars and other carbohydrates, and lipids.

The term "binding partner" refers to a molecule capable of specifically binding an analyte. The binding partner can be of several different types of molecules, including antibodies or antigen-binding fragments thereof or other proteins, peptides, polysaccharides, lipids, nucleic acids or nucleic acid analogs, such as oligonucleotides, aptamers, or PNAs (peptide nucleic acids). It may be any one.

The term "CD" refers to surface antigen cluster of differentiation.

The term "correction" in flow cytometry is a mathematical process that corrects for fluorescence extravasation (spectral overlap in multiparameter flow cytometry data). For example, correction may be performed by removing the signal of any given fluorochrome from all detectors except those specialized for measuring that dye. Because fluorescent dyes may have a wide spectrum, they may overlap and cause unwanted confusion during data analysis.

The term "labeled binding partner" refers to a binding partner that is conjugated to a dye. The term "reaction solution" refers to a solution containing a labeled binding partner. In some embodiments, in addition to the labeled binding partner, the reaction solution may further include stabilizers, salts, buffers, surfactants, and/or other reagents.

The term "linker" or "linkage" refers to a connecting moiety that connects two groups and has a backbone of 100 atoms or less in length. A linker or linkage is a chain of between 1 and 100 atoms in length, e.g., 1, 2, 3, 4, 5, 6 in length, even if it is a covalent bond connecting two groups. , 8, 10, 12, 14, 16, 18, 20, or more carbon atoms, and the linker may be linear, branched, cyclic, or single atom. It's okay. In some embodiments, the linker is a branched linker that refers to a binding moiety that connects three or more groups. In certain cases, one, two, three, four, or five or more carbon atoms of the linker backbone are optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. Good too. In some embodiments, the linker backbone includes a binding functional group, such as an ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. Bonds between backbone atoms may be saturated or unsaturated, and in some cases no more than one, two, or three unsaturated bonds are present in the linker backbone. The linker can be one or more substituents with, for example, alkyl, aryl or alkenyl groups. Linkers include, but are not limited to, polyethylene glycols, ethers, thioethers, tertiary amines, alkyls that may be linear or branched, such as methyl, ethyl, n-propyl, -methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc. The linker backbone can be a cyclic group, such as an aryl, heterocycle, or cycloalkyl group, in which two or more atoms, e.g. 2, 3 or 4 atoms of the cyclic group are present within the backbone. include. Linkers can be cleavable or non-cleavable.

The linker moiety may be attached to "A" as taught in U.S. Published Application No. 2020/0190253A1, which is incorporated herein by reference in its entirety, or 2019/0144601, which application is hereby incorporated by reference in its entirety. The linker moiety may include a sulfonamide, disulfonamide, selenomide, sulfinamide, sultam, disulfinamide, amide, seleninamide, phosphonamide, phosphinamide, phosphonamidate, or secondary amine.

As described therein, the term "sulfonamide" refers to the -S(O) 2 NR- moiety; the term "disulfonamide" refers to the -S(O) ₂ NR- moiety, each as related to the linker moiety; (O) ₂ NRS(O) ₂ - moiety; the term "selenone amide" refers to the -Se(O) ₂ NR- moiety; the term "sulfinamide" refers to the -S(O)NR- moiety; The term "disulfinamide" refers to the -S(O)NRS(O)- moiety; the term "seleninamide" refers to the -Se(O)NR- moiety; the term "phosphonamide" refers to the -S(O)NRS(O)- moiety; NR-PR(O)NR- moiety; the term "phosphinamide" refers to the -PR(O)NR- moiety; the term "phosphonamidate" refers to the -O-PR(O)NR- moiety the term "sultam" refers to a cyclic sulfonamide in which, for example, the R group is attached to the sulfur atom through an alkylene moiety; for each term, the R group independently , H, alkyl, haloalkyl, or aryl.

The term "terminal" as used herein refers to the terminus of a conjugate polymer chain that can include a functional group that provides bioconjugation. In some cases, such functional groups are referred to as terminal linkers. The terminal end is, for example, hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate. , boronic acid substituted aryl, boronic ester substituted aryl, boronic ester, boronic acid, optionally substituted tetrahydropyrene (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP); conjugated to amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazine, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and substrates or binders. It may also be an aryl or heteroaryl substituted with one or more pendant chains terminating with a functional group selected from those protecting groups.

The term "MdFI" or "MDFI" refers to the median fluorescence intensity.

The term "% recruitment" refers to the number of gated cells in the appropriate population.

The term "multiplex" as used herein refers to an assay or other analytical method in which multiple analytes can be assayed simultaneously.

The term "PEG" refers to polyethylene glycol or poly(ethylene glycol). The number after "PEG" refers to average molecular weight, Mw refers to weight average molecular weight, and Mn refers to number average molecular weight.

The term "PBS" refers to phosphate buffered saline, which is an aqueous buffer that may include sodium chloride, disodium hydrogen phosphate, potassium chloride, and potassium dihydrogen phosphate. For example, PBS may contain milliQ water or deionized water and 137mM NaCl, 2.7mM KCl, _{10mM Na2HPO4} , _{1.8mM KH2PO4} . The pH may be about pH 7.0-7.4. PBS may or may not be stored with an azide, such as sodium azide. PBS is an isotonic solution.

The acronym "SSC" refers to side scatter.

The acronym "WBC" refers to white blood cells.

A "dye" is a moiety that provides a detectable signal and may be directly or indirectly attached to or incorporated into a binding partner. The dyes used in this disclosure may be colored, fluorescent, or luminescent and are typically detected by a detector in a flow cytometer, such as a PMT or APD. Fluorescent dyes can be monomeric or polymeric. The fluorescent dye may be a fluorescent polymeric dye. Polymeric dyes are particularly useful in the analysis of chemical and biological targets. They are highly responsive optical reporters and efficient light absorbers because they contain multiple chromophores. Fluorescent polymeric dyes suitable for use in the present disclosure are described herein, for example in US 2019/0144601 and US 2020/0190253. Examples of polymeric dyes include, but are not limited to, conjugated polymers with repeating chromophore units, aggregates of conjugated molecules, and luminescent dyes attached via side chains to saturated polymers. , semiconductor quantum dots, and dendritic structures. The polymeric and monomeric dyes disclosed in U.S. Pat. Nos. 7,214,489, 8,354,239, and 8,575,303 may also be used in the present invention.

As used herein, the term "ammonium" refers to a cation having the formula _NHR3 ⁺ , where each R group is independently hydrogen, or substituted or unsubstituted alkyl, aryl, It is an aralkyl or alkoxy group. Preferably each R group is hydrogen.

As used herein, "oligoether" is understood to mean an oligomer containing structural repeating units with ether functionality. As used herein, "oligomer" is understood to mean a molecule containing one or more identifiable structural repeating units of the same or different formula.

The term "sulfonate functionality" or "sulfonate" as used herein refers to both the free sulfonate anion (-S(=O) ₂ O-) and its salts. Thus, the term sulfonate includes sulfonate salts such as sodium sulfonate, lithium sulfonate, potassium sulfonate, and ammonium sulfonate.

The term "sulfonamide" as used herein refers to a group of the formula -SO ₂ NR-, where R is hydrogen, alkyl, or aryl.

The term "alkyl" as used herein refers to a straight or branched saturated aliphatic radical having the indicated number of carbon atoms. For example, C ₁ -C ₆ alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, and the like. Alkyl can be any number of, for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2 May contain up to 4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6, and 5-6 carbons. Alkyl groups are typically monovalent, but may also be divalent, for example, if the alkyl group joins two moieties together.

The term "cycloalkyl" as used herein refers to a saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic ring containing from 3 to 12 ring atoms. Monocyclic rings referring to assemblies or indicating the number of atoms include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and polycyclic rings include, for example, norbornane, decahydronaphthalene, and adamantane. For example, C _3-8 cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

The term "haloalkyl" as used herein refers to alkyl as defined above in which some or all of the hydrogen atoms are replaced with halogen atoms. Halogen (halo) preferably represents chloro or fluoro, but may also be bromo or iodo. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluoro-phenyl, and the like. The term "perfluoro" defines a compound or radical in which at least two available hydrogens are replaced with fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy. .

As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

The term "alkoxy" as used herein refers to an alkyl group as defined above having an oxygen atom connecting the alkyl group to the point of attachment. Examples of alkoxy groups include methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy and the like. Alkoxy groups may be further substituted with various substituents described within. For example, an alkoxy group may be substituted with a halogen to form a "halo-alkoxy" group.

The term "alkene" as used herein refers to a straight or branched hydrocarbon having at least one double bond. Examples of alkene groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3 -pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3, 5-hexatrienyl. Alkene groups are typically monovalent, but may also be divalent, for example, if the alkenyl group links two moieties together.

The term "alkyne" as used herein refers to a straight or branched hydrocarbon having at least one triple bond. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3 -pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3, 5-hexatriynyl. Alkynyl groups are typically monovalent, but may also be divalent, for example, if the alkynyl group links two moieties together.

The term "aryl" as used herein refers to a monocyclic or fused bicyclic, tricyclic, or more cyclic aromatic ring containing from 6 to 16 ring carbon atoms. Refers to assembly. For example, aryl may be phenyl, benzyl, or naphthyl, preferably phenyl. "Arylene" means a divalent radical derived from an aryl group. Aryl groups include alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy, and oxy-C ₂ -C ₃ -alkylene; all of which are defined, for example, above. or mono-, di-, or by one, two, or three radicals selected from 1- or 2-naphthyl; or 1- or 2-phenanthrenyl; It may be substituted. Alkylenedioxy is a divalent substituent attached to two adjacent carbon atoms of phenyl, such as methylenedioxy or ethylenedioxy. Oxy-C ₂ -C ₃ -alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, such as oxyethylene or oxypropylene. An example of oxy-C ₂ -C ₃ -alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

Preferred as aryl are naphthyl, phenyl or phenyl mono- or di-substituted by alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenyl or mono- or di-substituted by alkoxy, halogen or trifluoromethyl. Disubstituted phenyl, especially phenyl.

The term "aryloxy" as used herein refers to the group O-aryl, where aryl is as defined above. Aryloxy groups may be unsubstituted or substituted with one or two suitable substituents. The term "phenoxy" refers to an aryloxy group in which the aryl portion is a phenyl ring. The term "heteroaryloxy" as used herein refers to the group -O-heteroaryl, where heteroaryl is as defined below. The term "(hetero)aryloxy" is used to indicate that the moiety is an aryloxy or heteroaryloxy group.

The term "polyethylene glycol" or "PEG" as used herein refers to a biocompatible compound based on ethylene glycol monomer units described by the formula -(CH2-CH2-O-) _n - or derivatives thereof. Refers to a family of water-soluble linear polymers. In some embodiments, "n" is 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less. less than 15 or less, such as from 3 to 15, or from 10 to 15. The PEG polymer groups may be of any convenient length, including, but not limited to, alkyl, aryl, hydroxyl, amino, acyl, carboxylic acid, carboxylate ester, acyloxy, and amide terminated and/or It is to be understood that various terminal groups and/or additional substituents may be included, including substituents.

The term "heteroaryl" as used herein means 1 to 4 of the ring atoms are heteroatoms, such as N, O, or S, and 5 to 16 ring atoms refers to monocyclic, or fused bicyclic or tricyclic aromatic ring assemblies that include. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or, for example, alkyl , nitro, or any other radicals substituted by halogen, especially mono- or di-substituted. Pyridyl represents 2-, 3- or 4-pyridyl, preferably 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinyl preferably represents 2-, 3- or 4-quinolinyl. Isoquinolinyl preferably represents 1-, 3- or 4-isoquinolinyl. Benzopyranyl and benzothiopyranyl each preferably represent 3-benzopyranyl or 3-benzothiopyranyl. Thiazolyl preferably represents 2- or 4-thiazolyl, most preferably 4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or substituted, especially Either a mono- or di-substituted radical.

Similarly, the substituents for aryl and heteroaryl groups can vary, with numbers ranging from zero to the total number of open valences on the aromatic ring system, including -halogen, -OR', -OC (O)R', -NR'R", -SR', -R', -CN, -NO ₂ , -CO ₂ R', -CONR'R", -C(O)R', -OC( O)NR'R", -NR"C(O)R', -NR"C(O) ₂ R', -NR'-C(O)NR"R''', -NH-C( _NH2 )=NH, -NR'C(NH ₂ )=NH, -NH-C(NH ₂ )=NR', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R'', -N ₃ , -CH(Ph) ₂ , perfluoro(C ₁ -C ₄ )alkoxy, and perfluoro(C ₁ -C ₄ )alkyl; R''' is hydrogen, (C ₁ -C ₅ )alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C ₁ -C ₄ )alkyl, and (unsubstituted aryl)oxy- independently selected from (C ₁ -C ₄ )alkyl.

Two of the substituents on atoms adjacent to the aryl or heteroaryl ring may be optionally replaced with substituents of the formula -TC(O)-(CH ₂ ) _q -U- , where T and U are independently -NH-, -O-, -CH ₂ -, or a single bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on atoms adjacent to the aryl or heteroaryl ring may be optionally replaced with substituents of the formula -A-(CH ₂ ) _r -B-, in which , A and B are independently -CH ₂ -, -O-, -NH-, -S-, -S(O)-, -S(O) ₂ -, -S(O) ₂ NR' -, or a single bond, and r is an integer from 1 to 3. One of the single bonds of the new ring thus formed may optionally be replaced by a double bond. Alternatively, two of the substituents on atoms adjacent to the aryl or heteroaryl ring are optionally replaced with substituents of the formula -(CH ₂ ) _s -X-(CH ₂ ) _t - Often, where s and t are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O) ₂ - or -S(O) ₂ NR'-. The substituent R' in -NR'- and -S(O) ₂ NR'- is selected from hydrogen or unsubstituted (C ₁ -C ₆ ) alkyl.

The term "(hetero)arylamino" as used herein refers to an amine radical substituted with an aryl group (eg, -NH-aryl). Arylamino may also be an aryl radical substituted with an amine group (eg -aryl-NH ₂ ). Arylamino may be substituted or unsubstituted.

The term "amine" as used herein refers to an alkyl group, as defined within, having one or more amino groups. Amino groups may be primary, secondary, or tertiary. Alkylamines may be further substituted with hydroxy groups. Amines useful in the present invention include, but are not limited to, ethylamine, propylamine, isopropylamine, ethylenediamine, and ethanolamine. The amino group is bonded together with at least two carbon atoms of the alkyl group, whether attached to the point of attachment of the alkylamine to the rest of the compound or present in the omega position of the alkyl group. Good too. Those skilled in the art will appreciate that other alkylamines are useful in the present invention.

The term "carbamate" as used herein refers to a functional group having the structure -NR"CO ₂ R', where R' and R" are hydrogen, (C ₁ -C ₈ ) independently selected from alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C ₁ -C ₄ )alkyl, and (unsubstituted aryl)oxy-(C ₁ -C ₄ )alkyl . Examples of carbamates include t-Boc, Fmoc, benzyloxy-carbonyl, alloc, methyl carbamate, ethyl carbamate, 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluor Olenyl methyl carbamate, Tbfmoc, Climoc, Bimoc, DBD-Tmoc, Bsmoc, Troc, Teoc, 2-phenylethyl carbamate, Adpoc, 2-chloroethyl carbamate, 1,1-dimethyl-2-haloethyl carbamate, DB-t -BOC, TCBOC, Bpoc, t-Bumeoc, Pyoc, Bnpeoc, V-(2-pivaloylamino)-1,1-dimethylethylcarbamate, and NpSSPeoc.

The term "carboxylate" as used herein refers to the conjugate base of a carboxylic acid and can be generally represented by the formula RCOO. For example, the term "magnesium carboxylate" refers to a magnesium salt of a carboxylic acid.

The term "activated ester" as used herein refers to a carboxyl activating group used in peptide chemistry to facilitate the facile condensation of a carboxyl group with a free amino group of an amino acid derivative. Descriptions of these carboxyl activating groups are commonly found in textbooks of peptide chemistry; for example, K. D. Kopple, "Peptides and Amino Acids", W. A. Benjamin, Inc., New York, 1966, pp. 50-51, and E. Found in Schroder and K. Lubke, "The Peptides"; Vol. 1, Academic Press, New York, 1965, pp. 77-128.

The terms "hydrazine" and "hydrazide" refer to compounds containing single bonded nitrogens, one of which is a primary amine functionality.

The term "aldehyde" as used herein refers to a chemical compound having a -CHO group.

The term "thiol" as used herein refers to a compound that contains a functional group comprised of sulfur-hydrogen bonds. The general chemical structure of the thiol functional group is R-SH, where R represents an alkyl, alkene, aryl, or other carbon-containing group.

The term "silyl" as used herein refers to Si( ^Rz ) ₃ , where each ^Rz is independently alkylaryl, or other carbon-containing group.

The term "diazonium salt" as used herein refers to a group of organic compounds having the structure ^RN ₂ ⁺ aryl), and X is an inorganic or organic anion (eg, halogen).

The term "triflate", also referred to as trifluoromethanesulfonate, is _a group with the formula _CF3SO3 .

The term "boronic acid" as used herein refers to the structure -B(OH) ₂ . Those skilled in the art will recognize that the boronic acid may be present as a boronate ester at various stages in the synthesis of the quencher. Boronic acid is meant to include such esters. The term "boronate ester" or "boronate ester" as used herein refers to a chemical compound containing a -B(Z ¹ )(Z ² ) moiety, where Z ¹ and Z ² together form the moiety and the atom bonded to the boron is in each case an oxygen atom. The boronic ester moiety may be a 5-membered ring. The boronic ester moiety may be a 6-membered ring. The boronic ester moiety may be a mixture of 5- and 6-membered rings.

Values expressed in range format include not only the numbers specifically listed as the limits of the range, but also each number and every individual number subsumed within that range, as if the subrange were specifically listed. or should be interpreted in a flexible manner to include subranges. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" does not mean only about 0.1% to about 5%; 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) ) should be construed as including. The phrase "from about X to Y" has the same meaning as "from about X to about Y" unless otherwise specified. Similarly, the phrase "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless specified otherwise.

As used herein, "dye conjugate" refers to a binding partner that is conjugated to a non-polymeric or polymeric dye.

As used herein, "SN" refers to Beckman Coulter, Inc. ``Super Nova'' dye commercially available from.

As used herein, “EMPIGEN BB®” refers to a zwitterionic surfactant ( CAS number 66455-29-6).

The term "about" as used herein refers to the degree of variation in a value or range, such as within 10%, 5%, or 1% of a stated value or of the limits of a stated range. It is possible to accept less than

As used herein, "specific binding" refers to the binding of an antibody or other binding partner (e.g., in a polymer-conjugated dye) to an epitope of a cell or target analyte that the antibody or binding partner targets. Refers to a combination.

As used herein, "non-specific binding" refers to binding of an antibody or other binding partner (e.g., in a polymer-conjugated dye) to cells that do not contain the epitope that the antibody or other binding partner targets. Refers to binding to sample components. For example, non-specific binding occurs when an antibody binds to a cell that does not have an epitope specific for the antibody.

As used herein, "reducing" or "eliminating" nonspecific binding of a polymer dye conjugate refers to "negative" (e.g., negative granulocyte, monocyte, and lymphocyte populations). ) has a mean fluorescence intensity (MFI) of at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least 99%, or more; about 50% to about 95%, about 50% to about 75%, about 60% to about 80%, or about 65% to about 90% ) may also refer to the case where it decreases. In other words, the % reduction in at least one of the background staining of monocytes, granulocytes, and lymphocytes is at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least 99%, or more; about 50% to about 95%, about 50% to about 75%; from about 60% to about 80%, or from about 65% to about 90%).

The term "substantial" or "substantially" as used herein means at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, Refers to a majority or majority, such as 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999%, or greater.

The terms "substantially free" or "substantially free" as used herein mean less than about 1%, less than 0.5%, less than 0.1%, less than 0.05%, at less than about 0.0005% or less; at less than about 0.0005% or less; at less than about 0.0005% or less; at less than about 0.0005% or less; Point.

In this document, the terms "a," "an," or "the" include one or more than one, unless the context clearly dictates otherwise. used as. The term "or" is used to refer to a non-exclusive "or" unless specified otherwise. Furthermore, it is to be understood that any terminology or terminology used herein, and not specifically defined, is for purposes of explanation only and not as limitation. Any use of section headings is intended to facilitate reading this document and should not be construed as limiting. Additionally, information related to a section heading may reside within or outside of that particular section. Additionally, all publications, patents, and patent documents mentioned in this document are herein incorporated by reference in their entirety as if individually incorporated by reference. In the event of a discrepancy in usage between this document and a document so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for unresolvable discrepancies, Preference is given to use in this document.

In certain embodiments, dye compositions are provided that include at least one polymeric dye conjugate and at least one suitable zwitterionic surfactant.

In certain embodiments, dye compositions are provided that include at least one polymeric dye conjugate and at least one suitable anionic surfactant.

In some embodiments, a method for reducing or eliminating nonspecific binding of at least one polymeric dye conjugate to cells in a biological sample, e.g., a blood sample, the method comprising: at least one polymeric dye conjugate and at least one zwitterionic and/or anionic surfactant before, during, and/or after contacting the dye conjugate with the biological sample. A method is provided that includes contacting. Steps may be performed in any order without departing from the principles of the invention, unless explicitly recited in temporal or operational order. Furthermore, certain steps may be performed simultaneously, unless explicit claim language dictates that they be performed separately. For example, the steps of doing X as claimed in the claims and the steps of doing Y as claimed in the claims may be performed simultaneously within one operation, and the resulting process is , are within the literal scope of the claimed process. Thus, in some cases, the at least one polymeric dye conjugate is contacted with at least one zwitterionic or anionic surfactant prior to contacting the at least one polymeric dye conjugate with the blood sample. Good too. In some cases, the at least one polymeric dye conjugate may be contacted with at least one zwitterionic or anionic surfactant at the same time that the at least one polymeric dye conjugate is contacted with the blood sample. .
surfactant

Various types of surfactants have been investigated to reduce or prevent non-specific interactions of polymer dye conjugates with biological samples.

Suitable surfactants may be zwitterionic surfactants or certain anionic surfactants. Examples of suitable surfactants include the general formula

R ^1' [CO-X(CH ₂ ) _j ] _g - [N ⁺ (R ^2' ) (R ^3' )] _k - (CH ₂ ) _f - [CH(OH)CH ₂ ] _h -Y ^- ( where R ^1' is saturated or unsaturated C _5-24 alkyl, such as C _6-22 , C _5-21 , C _7-19 , C _11-17 , or C _8-18 alkyl, saturated C _{10- 16} alkyl, or saturated C _12-14 alkyl; X is NH, NR ⁴ ′, where R ^4′ is C _1-4 alkyl, O, or S; an integer of 10, such as 2 to 5 and 3; g is 0 or 1; R ^2' and R ^3' are each independently C _1-4 alkyl, such as ethyl or methyl; hydroxy is optionally substituted with a hydroxyethyl group or methyl; k is 0 or 1; f is an integer from 0 to 4, such as 0, 1, 2, 3, or 4; h is 0 or 1; Y is COO, SO ₃ , OPO(OR ^5′ )O, or P(O)(OR ^5′ )O, where R ^5′ is H or C _{1 ~4} alkyl), and when k=0, the surfactant can be in acidic form or its sodium or potassium salt.

Surfactants can be present in buffers or other suitable aqueous compositions according to the present disclosure at about 0.05% to about 0.25%, about 0.06% to about 0.2%, or about 0.08%. and about 0.16% (w/v).

Suitable zwitterionic surfactants that may be used according to the methods described herein include betaine zwitterionic surfactants, such as alkylbetaines, alkylamidobetaines, amidazolinium betaines, sulfobetaines. (INCI Sultaine), as well as phosphobetaine.

Examples of suitable zwitterionic surfactants include alkyl betaines, such as those of the formula:
R ^1' -N ⁺ (CH ₃ ) ₂ -CH ₂ COO ^- ;
R ^1' -CO-NH(CH ₂ ) ₃ -N ⁺ (CH ₃ ) ₂ -CH ₂ COO ^- ;
R ^1' -N ⁺ (CH ₃ ) ₂ -CH ₂ CH(OH)CH ₂ SO ₃ ^- ; and R ^1' -CO-NH- (CH ₂ ) ₃ -N ⁺ (CH ₃ ) ₂ -CH ₂ CH (OH)CH ₂ SO ₃ ^-
There is something.

Examples of suitable betaines and sulfobetaines are the following (written according to INCI): almond amidopropyl betaine, apricot amidopropyl betaine, avocado amidopropyl betaine, babasamidopropyl betaine, behenamidopropyl betaine, behenyl betaine, ca Nolamidopropyl betaine, caprylic/capramidopropyl betaine, carnitine, cetyl betaine, cocamidoethyl betaine, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, coco betaine, coco hydroxysultaine, coco/oleamidopropyl betaine, coco sultaine , decyl betaine, dihydroxyethyl oleyl glycinate, dihydroxyethyl soybean glycinate, dihydroxyethyl stearyl glycinate, dihydroxyethyl tallow glycinate, PG-betaine dimethicone propyl, dorcamidopropyl hydroxysultaine, hydrogenated tallow betaine, isostearamidopropyl Betaine, lauramidopropyl betaine, lauryl betaine, lauryl hydroxysultaine, lauryl sultaine, milkamidopropyl betaine, milkamidopropyl betaine, myristamidopropyl betaine, myristyl betaine, oleamide propyl betaine, oleyl hydroxysultaine, oleyl Betaine, olive amidopropyl betaine, palmamidopropyl betaine, palmitamidopropyl betaine, palmitoyl carnitine, palm kernel amidopropyl betaine, polytetrafluoroethylene acetoxypropyl betaine, ricinolamide propyl betaine, sesamidopropyl betaine, soy amidopropyl betaine, They are stearamidopropyl betaine, stearyl betaine, tallow amidopropyl betaine, tallow amidopropyl hydroxysultaine, tallow betaine, tallow dihydroxyethyl betaine, undecylenamidopropyl betaine and wheat germ amidopropyl betaine.

Suitable betaine zwitterionic surfactants include N-(alkyl C _10-16 )-N,N-dimethylglycine betaine, N-(alkyl C _12-14 )-N,N-dimethylglycine betaine, N, Whether it is N-dimethyl-N-dodecylglycine betaine, lauryl dimethyl betaine (also known as lauryl betaine), myristyl sulfobetaine, or n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate good. Lauryl betaine is commercially available as EMPIGEN BB® (Huntsman Corporation) and has a CMC (20-25° C.) of 1.6-2.1 mM. Myristylsulfobetaine (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, also known as DMMA) is a trademark of ZWITTERGENT® 3-14 (Merck KGaA, Darmstadt, Germany). It has a CMC of 100-400 uM. n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (3-N,N-dimethylpalmitylammonio)propanesulfonate, also known as DMPA) is ZWITTERGENT® 3- It is available under 16 trade names and has a CMC of 10-60 uM. For example, coconut dimethyl betaine is commercially available from Seppic under the trade name AMONYL265®; lauryl betaine is commercially available from Sigma-Aldrich under the trade name EMPIGEN BB®. A further example of a betaine is lauryl-imino-dipropionate, commercially available from Rhodia under the tradename MIRATAINE H2C-HA®.

The zwitterionic surfactant is present in the buffer or other suitable aqueous composition according to the present disclosure in the range of about 0.06% to about 0.2% or about 0.08% to about 0.16%. May exist.

Examples of suitable anionic surfactants include sarcosinate surfactants in acidic or neutral form. For example, a suitable anionic surfactant may be a neutral form of a sarcosinate surfactant. The sarcosinate surfactant may be an alkanoyl sarcosinate surfactant.

Examples of suitable anionic surfactants include those having the general formula R ^1' [CO-X(CH ₂ ) _j ] _g -(CH ₂ ) _f -[CH(OH)CH ₂ ] _h -Y ^- , R ^1′ is saturated or unsaturated C _5-24 alkyl, such as C _8-18 alkyl, saturated C _10-16 alkyl, or saturated C _12-14 alkyl; X is NH, NR ⁴ ′ , where R ^4' is C _1-4 alkyl, O, or S; j is an integer from 1 to 10, such as 2 to 5 and 3; g is 0 or 1; f is an integer from 0 to 4, such as 0, 1, 2, 3, or 4; h is 0 or 1; Y is COO, _SO3 , OPO(OR5 ^' )O, or P(O)(OR ^5' )O, where R ^5' is H or C _1-4 alkyl); anionic surfactants are in the acidic form, or It may also be in the form of sodium or potassium salts.

Suitable anionic surfactants have the structure CH ₃ (CH ₂ ) _a CH ₂ (CH ₂ CH=CH) _b CH ₂ (CH ₂ ) _c CH ₂ (C=O)N(CH ₃ )CH ₂ CO ₂ It may contain X (in the formula, a=1 to 8; b=0 to 2; c=0 to 6; and X=H, Na, K).

Examples of alkanoyl sarcosinates include the formula:
R ^1' -CO-N(CH ₃ )-CH ₂ -COO ^- ; and R ^1' -CO-N(CH ₃ )-CH ₂ -SO ₃ ^- (where R ^1' is saturated or unsaturated C _5-24 alkyl, C _7-19 alkyl, or C _11-17 alkyl), and, for example, their sodium or potassium salts.

Examples of suitable alkanoyl sarcosinates and their acid or salt forms include N-lauroyl sarcosine, sodium lauroyl sarcosinate, sodium palmitoyl sarcosinate, sodium stearoyl sarcosinate, N-methyl-N-(1-oxotetra decyl)-glycine sodium salt, sodium caproyl sarcosinate, sodium capryloyl sarcosinate, N-methyl-N-(1-oxo-9-octadecen-1-yl)-glycine, sodium salt, sodium oleoyl sarcosinate, and sodium linoleoyl sarcosinate.

The anionic surfactant is present in the buffer or other suitable aqueous composition according to the present disclosure in a range of about 0.06% to about 0.2% or about 0.08% to about 0.16%. You can leave it there.

The compositions can be used in flow cytometry and thus include any suitable carrier, stabilizer, buffer, salt, chelating agent (e.g., EDTA), or preservative. Additional components may be included, including one or more of: The composition may also include one or more additional surfactants in addition to the zwitterionic and/or anionic surfactants described herein. Non-limiting examples of one or more additional surfactants include polysorbates, such as TWEEN® 20 (polyoxyethylene sorbitan monolaurate) and TWEEN® 80 (polyoxyethylene sorbitan monolaurate). monooleate). The carrier can be an aqueous solution, such as water, saline, alcohol, or a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. The carrier may also include formulation agents, such as suspending, stabilizing, and/or dispersing agents. The composition may also include a buffer or pH adjusting agent; typically a buffer is a salt prepared from an organic acid or base. Typical buffering agents include salts of organic acids such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; tristromethamine hydrochloride, or phosphate buffers. .

The composition may include a protein stabilizer selected from the group consisting of bovine serum albumin (BSA or "fraction V"), casein, and gelatin. The protein stabilizer may be BSA, a commercially available bovine serum albumin protein of bovine origin. The protein stabilizer is present in the buffer or other suitable aqueous composition according to the present disclosure at about 0.1-5 mg/mL, about 0.5-3 mg/mL, or about 2 mg/mL. Good too.

The stabilizer may be a gelatin, a protein, and is usually derived from collagen obtained from an animal body part. It is brittle when dry and rubbery when wet. After undergoing hydrolysis, it may also be referred to as hydrolyzed collagen, collagen hydrolyzate, gelatin hydrolyzate, hydrolyzed gelatin, and collagen peptide. Several types of gelatin are commercially available, including gelatin type A, gelatin type B, Prionex® highly purified gelatin type A, and cold water fish gelatin.

The composition may also include any suitable preservative. Preservatives may be antioxidants, biocides, or antimicrobials. Preservatives may be inorganic salts. The preservative may be sodium azide. Preservatives may be present in a concentration range of about 0.01 to about 1%, about 0.05% to about 0.5%, or about 0.1%.
polymer dye

In another embodiment, the composition may be used with polymeric dyes. The polymeric dye may be a fluorescent polymeric dye or a fluorescent polymeric tandem dye. Polymeric dyes are particularly useful in the analysis of chemical and biological target analytes. They are highly responsive optical reporters and efficient light absorbers because they contain multiple chromophores. The polymer dye conjugate may include any previously disclosed fluorescent polymer dye or fluorescent polymer tandem dye.

For example, polymer dyes or tandem polymer dyes can be used in published PCT applications WO2017/180998; US Application No. 2021/0047476; US Application No. 2020/0190253; Application No. 2018/0224460; U.S. Patent No. 11,034,840; U.S. Patent No. 10,228,375; U.S. Patent No. 10,545,137B2; U.S. Patent No. 10,533,092; U.S. Patent No. No. 7,214,489; U.S. Pat. No. 8,354,239; U.S. Pat. No. 8,575,303, each of which is incorporated herein in its entirety. is incorporated by reference as if fully set forth in . The polymeric dye conjugate may have the structure of any of the water-soluble fluorescent polymeric dyes disclosed in published U.S. Application No. 2020/0190253A1, which application is incorporated herein in its entirety. Incorporated by reference as if mentioned. The polymeric dye conjugate may have the structure of any of the water-soluble fluorescent polymeric dyes disclosed in published U.S. Application No. 2019/0144601, which application is incorporated herein in its entirety. Incorporated by reference as if mentioned.

The polymeric dye or polymeric dye conjugate can be any commercially available polymeric dye or polymeric dye conjugated to a binding partner. The polymer dye or polymer dye conjugate may include a polymer dye excitable by a violet laser. The polymeric dye or polymeric dye conjugate may include a polymeric dye excitable by a violet laser, for example at 405 nm. The polymer dye or polymer dye conjugate may include a violet laser (405 nm) excitable polymer dye.

In some embodiments, the polymeric dye or polymeric dye conjugate may include SuperNova™ dye (Beckman Coulter, Inc.). SuperNova™ polymers are a new generation of polymeric dyes useful for flow cytometry applications. The polymer dye or polymer dye conjugate may include SuperNova™ v428, SuperNova™ v605, or SuperNova™ v786 (Beckman Coulter, Inc.). SuperNova™ v428 has unique photophysical properties that result in extremely bright conjugates when conjugated to antibodies or other binding partners. For example, SuperNova™ v428 (SN v428) (Beckman Coulter, Inc.) is a polymeric dye that is optimally excited by a violet laser (e.g., 405 nm), with a maximum excitation wavelength of 414 nm and an emission peak of 428 nm. and can be detected using a 450/50 bandpass filter or equivalent.

SuperNova™ v428 is one of the brightest dyes excitable by a violet laser and is therefore particularly suitable for evaluating dimly expressed markers. SuperNova™ conjugate antibodies include anti-CD19 antibody-SuperNova™ v428, anti-CD22 antibody-SuperNova v428, anti-CD25 antibody-SuperNova™ v428, and anti-CD38 antibody-SuperNova™ v428 antibody-polymer. Possible dye conjugates.

SuperNova™ v605 and SuperNova™ v786 (Beckman Coulter, Inc.) are tandem polymer dyes derived from the core of SuperNova™ v428 polymer dye. Both share the same absorbance properties, with a maximum excitation wavelength of 414 nm. The emission peaks of SuperNova™ v605 and SuperNova™ v786 are 605 nm and 786 nm, respectively, so they are best detected using the 610/20 and 780/60 nm bandpass filters of a flow cytometer. SuperNova™ v605 and SuperNova™ v786 may be conjugated with anti-CD19, anti-CD22, anti-CD25, and anti-CD38 antibodies, for example.

The polymeric dye or polymeric dye conjugate may include a polymeric dye excitable by an ultraviolet (“UV”) laser. The polymer dye or polymer dye conjugate comprises a polymer dye excitable by a UV laser at a wavelength of 320 nm to 380 nm, 340 nm to 360 nm, 345 nm to 356 nm, or less than or equal to 380 nm but greater than or equal to 320 nm. Good too. The polymer dye or polymer dye conjugate may include a UV excitable polymer dye. The UV excitable polymer dye or polymer dye conjugate may typically emit light at a wavelength of 380 nm to 430 nm, 406 nm to 415 nm, or less than or equal to 430 nm but greater than or equal to 380 nm.

The polymer dye or polymer dye conjugate is a Brilliant Violet™ dye (BioLegend®/Sirigen Group Ltd.), such as Brilliant Violet 421™ (excitation wavelength maximum 405 nm, maximum emission wavelength 421 nm, 450/50 filter ), Brilliant Violet 510(TM) (maximum excitation wavelength 405nm, maximum emission wavelength 510nm, 510/50 filter), Brilliant Violet 570(TM) (maximum excitation wavelength 405nm, maximum emission wavelength 570nm, 585/42 filter), Brilliant Viole t 605(TM) (maximum excitation wavelength 405 nm, maximum emission wavelength 603 nm, 610/20 filter), Brilliant Violet 650(TM) (maximum excitation wavelength 405 nm, maximum emission wavelength 645 nm, 660/20 filter), Brilliant Violet 711(TM) (maximum excitation wavelength 405 nm, maximum emission wavelength 711 nm, 710/50 filter), Brilliant Violet 750™ (maximum excitation wavelength 405 nm, maximum emission wavelength 750 nm, 780/60 filter), Brilliant Violet 785™ (maximum excitation wavelength 405 nm, maximum emission wavelength 785 nm, 780/60 filter). The polymeric dye or polymeric dye conjugate may include Spark Violet™ 538 (BioLegend, Inc.) (excitation maximum wavelength 405 nm, maximum emission wavelength 538 nm).

The polymeric dye or polymeric dye conjugate may include Super Bright dye (Invitrogen, ThermoFisher Scientific). Super Bright dye can be excited by a violet laser (405 nm). Super Bright dyes include Super Bright 436 (maximum excitation wavelength 414 nm, maximum emission wavelength 436 nm, 450/50 bandpass filter), Super Bright 600 (maximum emission wavelength 600 nm, 610/20 bandpass filter), Super Bright 645 (maximum emission wavelength 645 nm wavelength, 660/20 band pass filter), or Super Bright 702 (maximum emission wavelength 702 nm, 710/50 band pass filter).

The polymer dye or polymer dye conjugate may include BD Horizon Brilliant™ Violet polymer dye (Becton, Dickinson and Co., BD Life Sciences). Polymer dyes include BD Horizon Brilliant™ BV421 (450/40 or 431/28 filter), BV480 (525/40 filter), BV510 (525/40 filter), BV605 (610/20 filter), BV650 (660/28 filter). 20 filter), BV711 (710/50 filter), and BV786 (786/60 filter).

Polymeric dyes may be prepared synthetically by polymerization of monomers, thereby forming highly conjugated fluorescent backbones. Capping may be performed on the polymer by activation using appropriate functional groups, resulting in a polymer that can be conjugated to a binding partner. Alternatively, the polymer may be activated and conjugated by attaching appropriate functional groups on the polymer backbone. Activated polymers can be conjugated to binding partners. Any suitable binding partner may be used, eg, with an antibody, followed by purification, eg, by using standard procedures. Functional groups include amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazine, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and conjugates to substrates or binding partners. may be selected from the group consisting of those protecting groups for protecting.

The polymer dye conjugate may include a fluorescent polymer having monomer subunits including, but not limited to, dihydrophenanthrene (DHP), fluorene, and combinations thereof. In some embodiments, the polymeric dye conjugate has formula III:
It may contain a polymer dye having the structure.

Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers. Each A may be substituted with a functional group to be conjugated to a binding partner.

Each optional M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap modifying monomers, optionally substituted ethylene, and ethynylene and is uniform along the polymer backbone. or randomly distributed. Each M is
(wherein, each M is an amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, amide, sulfonamide) , ethers, thioethers, thiocarbamates, hydroxyls, iodoacetyl, hydrazides, hydrazinos, ketones, phosphines, epoxides, ureas, thioureas, thioesters, imines, disulfides, and conjugates to another substrate, acceptor dye, molecule, or binder optionally substituted and terminated with a functional group selected from those protecting groups for
Each R ⁵ is halogen, hydroxyl, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkene, C ₂ -C ₁₂ alkyne, C ₃ -C ₁₂ cycloalkyl, C ₁ -C ₁₂ haloalkyl, C ₁ -C ₁₂ Alkoxy, C ₂ -C ₁₈ (hetero)aryl group, C ₂ -C ₁₈ (hetero)aryloxy, C ₂ -C ₁₈ (hetero)arylamino, carboxylic acid, carboxylate ester, (CH ₂ ) _x' (OCH ₂ -CH ₂ ) _y' OCH ₃ , and (CH ₂ ) _x' (OCH ₂ -CH ₂ ) _y' OCF ₃ , where each x' is independently 0 to 20 is an integer of , and each y' is independently an integer of 0 to 50;
Each R ¹ is independently an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a moiety:
And;
Each R ² is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, Ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, or moieties
And;
each R ³ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups;
each Z is independently selected from the group consisting of C, O, and N;
each Q is independently selected from the group consisting of a bond, NH, ^NR4 , and _CH2 ;
each subscript n is independently an integer from 0 to 20)
may be independently selected from the group consisting of:

The linker is represented by formula III as L. Each optional linker L may be an aryl or heteroaryl group distributed uniformly or randomly along the polymer backbone, including amines, carbamates, carboxylic acids, carboxylates, maleimides, activated Terminated with a functional group selected from the group consisting of esters, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkynes, aldehydes, thiols, and protecting groups thereof for conjugation to substrates or binding partners. may be substituted with one or more pendant chains.

The polymer conjugates of the present disclosure also include termini represented by Formula III as each G ¹ and G ² . The terminus may be modified or unmodified. Terminals include hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boron Acid-substituted aryl, boronate-substituted aryl, boronic ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene; amine, carbamate, carboxylic acid, carboxylate, maleimide , activated esters, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkynes, aldehydes, thiols, and protecting groups thereof optionally conjugated to the substrate or binding partner. Each may be independently selected from the group consisting of aryl or heteroaryl substituted with one or more pendant chains terminating in .

In the structure of Formula III, a, c, and d define the mol% of each unit, each of which may be repeated uniformly or randomly, and each a represents a mol% of 10 to 100%. and each c is 0 to 90% mol%; each d is 0 to 25% mol%; each b is independently 0 or 1; each m is 1 is an integer of about 10,000.

In some embodiments, the polymeric dye conjugate has Formula I:
(In the formula:
each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers;
L ¹ , L ² , and L ³ are linker moieties;
W is a water-soluble moiety;
each E is an independently selected chromophore, functional moiety, or binding partner;
each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene, and ethynylene;
G ¹ and G ² are independently selected from unmodified polymer ends and modified polymer ends;
subscripts n and m are independently integers ranging from 1 to 10,000;
The subscript p is an integer ranging from 0 to 10,000;
The sum of subscripts n, m, and p ranges from 2 to 10,000;
subscript q is 1, 2, 3, or 4;
the subscript r is 1, 2, 3, or 4;
The subscript s is 0, 1, 2, or 3;
the subscript t is 1 or 2;
The sum of the subscripts r and s ranges from 1 to 4;
A and B are randomly or non-randomly distributed in the conjugate polymer)
It may have the structure.
L ¹ may be a sulfonamide, sulfonamide, sultam, disulfinamide, amide, phosphonamide, phosphonamidate, phosphinamide, or secondary amine. Alternatively, L ¹ may be a sulfonamide, amide, phosphonamide, or secondary amine.
Subscript q may be equal to the sum of subscripts r and s, subscript r may be 1 or 2, and if subscript r is 1 then subscript s is 0 or 1, and if subscript r is 2, subscript s is 0.
Each L ³ may be a covalent bond.

The conjugate polymer has formula II:
(In the formula:
L ^1a is a linker part;
R ¹ is selected from the group consisting of H and amine protecting groups)
It may have a structure according to.
A variety of linkers L ^1a and L ² as described herein can be used for the synthesis of polymers according to Formula I and Formula II. for example:
L ^1a is a covalent bond, C _1-8 alkylene, 2- to 8-membered heteroalkylene (e.g., divalent alkoxy linker), C _3-8 cycloalkylene, C _6-10 arylene, 5 to 12-membered heteroarylene, may be selected from the group consisting of 12-membered heterocyclylene, -NHC(O)L ^a -, -C(O)NHL ^a -, -C(O)L ^a -, and combinations thereof;
L ² is a covalent bond, C _1-8 alkylene, 2- to 8-membered heteroalkylene (e.g., divalent alkoxy linker), C _3-8 cycloalkylene, C _6-10 arylene, 5 to 12-membered heteroarylene, 12-membered heterocyclylene, -L ^b NHC(O)-, -L ^b C(O)NH-, -L ^b C(O)-, -C(O)NHL ^b -, -C(O)L ^b -, and combinations thereof;
L ^a and L ^b may be independently selected from the group consisting of C _1-8 alkylene and 2 to 8 membered heteroalkylene;
R ¹ may be selected from the group consisting of H and amine protecting groups.
Polymers according to formula II are provided, where:
L ^1a is a group consisting of a covalent bond, C _1-8 alkylene, 2- to 8-membered heteroalkylene, -NHC(O)L ^a -, -C(O)NHL ^a -, and -C(O)L ^a - selected from
L ² is a covalent bond, C _1-8 alkylene; 2 to 8-membered heteroalkylene, -L ^b NHC(O)-, -L ^b C(O)NH-, -L ^b C(O)-, -C selected from the group consisting of (O)NHL ^b -, and -C(O)L ^b -;
L ^a and L ^b are independently selected from the group consisting of C _1-8 alkylene and 2 to 8 membered heteroalkylene;
R ¹ is selected from the group consisting of H and amine protecting groups).
W may include one or more ethylene glycol monomers. Alternatively, W may include poly(ethylene glycol).
^L3 is a first point of attachment to a first ^L1 moiety (or a first ^L1a moiety); a second point of attachment to a second ^L1 moiety (or a second ^L1a moiety); and a trivalent arylalkyl moiety having a third point of attachment to the A monomer.

For example, the present disclosure provides formula VI:
(In the formula,
L ^1a is as defined above;
L ² is as defined above;
W is as defined above;
L ^3a is a group consisting of a covalent bond, C _1-8 alkylene, 2- to 8-membered heteroalkylene, -NHC(O)L ^a -, -C(O)NHL ^a -, and -C(O)L ^a - selected from;
L ^a is selected from the group consisting of C _1-8 alkylene and 2 to 8 membered heteroalkylene; the wavy line is the point of attachment to the monomer)
A conjugate polymer having two or more chromophores attached thereto is provided as shown in FIG.
Each A monomer in a polymer having the structure of Formula I, II, or III may be the same monomer. Each A monomer in a polymer having the structure of Formula I, II, or III may be a different monomer. A may be a fluorescent monomer. A may be a 9,10-phenanthrene dione monomer (for example, a dihydrophenanthrene (DHP) monomer), a fluorene monomer, or a fluorene oxepine monomer.

Monomer A in the polymer having the structure of formula I, II or III is a DHP-based monomer, for example:
(In the formula:
each X is independently C or Si;
each Y is independently CR ¹ R ² or SiR ¹ R ² ;
Each R ¹ is independently an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a moiety
And;
Each R ² is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, Ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, or moieties
And;
each R ³ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups;
each Z is independently selected from the group consisting of C, O, and N;
each Q is independently selected from the group consisting of a bond, NH, ^NR4 , and _CH2 ;
each subscript n is independently an integer from 0 to 20)
It may be.

R ¹ has the structure shown below:
(wherein Q is NH and each R ³ is a group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG group) each Z is independently selected from the group consisting of C, O, and N)
It may have.

DHP monomer has the structure:
(In the formula:
Each subscript f is independently an integer from 0 to 50;
Each subscript n is independently an integer from 0 to 20;
Each R ² is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, Ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, or moieties
And;
Each R ⁵ is independently H, C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl, C ₃ -C ₂₀ cycloalkyl, C ₁ -C ₂₀ haloalkyl, C ₁ - C ₂₀ alkoxy, C ₂ -C ₂₆ aryloxy, C ₂ -C ₂₆ heteroaryloxy, C ₂ -C ₂₆ arylamino, or C ₂ -C ₂₆ heteroarylamino;
each Z is independently selected from the group consisting of C, O, and N)
It may have.

DHP monomer has the structure:
(In the formula:
Each subscript f is independently an integer from 0 to 50;
Each subscript n is independently an integer from 0 to 20;
Each R ² is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, Ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, or moieties
And;
Each R ⁵ is independently H, C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl, C ₃ -C ₂₀ cycloalkyl, C ₁ -C ₂₀ haloalkyl, C ₁ - C ₂₀ alkoxy, C ₂ -C ₂₆ aryloxy, C ₂ -C ₂₆ heteroaryloxy, C ₂ -C ₂₆ arylamino, or C ₂ -C ₂₆ heteroarylamino;
each Z is independently selected from the group consisting of C, O, and N)
It may have.

Monomer A in the polymer having the structure of formula I, II, or III is a fluorene-based monomer, for example:
(wherein X, Z, R ¹ , R ² , R ⁵ , subscript n, and subscript f are as defined herein)
It may be.

R ¹ and R ² groups, such as ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, or the structure:
The moiety with can confer solubility in water/buffer. In some embodiments, for example, the polymer is greater than 10 mg/mL, greater than 15 mg/mL, greater than 20 mg/mL, greater than 25 mg/mL, greater than 30 mg/mL, greater than 35 mg/mL, 40 mg/mL. mL, greater than 45 mg/mL, greater than 50 mg/mL, greater than 60 mg/mL, greater than 70 mg/mL, greater than 80 mg/mL, greater than 90 mg/mL, or greater than 100 mg/mL. be.

Monomer A is also a crosslinking monomer. For example, the crosslinking monomer of the present invention includes:
(wherein X, Y, ^R2 , and ^R5 are as defined above)
There is.

Monomer A in the polymer having the structure of formula I, II, or III is an oxepine-based monomer (e.g., a fluorene oxepine-based monomer), for example:
(wherein X, R ¹ and R ² are as defined herein)
It may be.
tandem polymer dye

The polymer may have an acceptor dye attached to the backbone, which will provide for monitoring the emission of the acceptor dye attached to the backbone via energy transfer. Acceptor dyes useful in tandem polymer dyes include, for example, FITC, CY3B, Cy55, Alexa 488, Texas red, Cy5, Cy7, Alexa 750, and 800CW. For example, the acceptor dye can be a linker L:
It may be bonded to the polymer via.
As described in U.S. Published Application No. 2020/0190253, which is incorporated herein by reference in its entirety, the acceptor dye is attached directly to monomer A as group E in the structure of Figure I or II above. You can also do that. The SuperNova tandem dyes SuperNova v605 and SuperNova v786 (Beckman Coulter, Inc.) are tandem polymer dyes derived from the core SuperNova v428. Both SuperNova v605 and SuperNova v786 share the same absorbance characteristics, with a maximum excitation wavelength of 414 nm. These are best detected using 610/20 and 780/60 nm bandpass filters on a flow cytometer, as the emission peaks are at 605 nm and 786 nm, respectively.
conjugate dye

Polymeric dyes may be conjugated to binding partners of different specificity, e.g., target analyte-specific antibodies, to synthesize binding partner-dye conjugates, e.g., CD19-SN v428, CD20-SN v605, etc. .

Polymeric dyes and polymeric dye conjugates may be formulated with aqueous buffers. Any suitable aqueous buffer may be used, such as an isotonic aqueous buffer, such as PBS buffer. There may be additives as aqueous buffers. For example, aqueous buffers include BSA, sodium azide, nonionic surfactants such as PF-68, and zwitterionic surfactants such as Empigen BB®, as described herein. ), or anionic surfactants such as NLS. BSA helps stabilize the conjugate, sodium azide prevents any microbial contamination, and surfactants, such as Empigen BB®, significantly reduce non-specific binding to monocytes and granulocytes. or remove. BSA may be present in a range of 0-3 mg/mL, 0.5-2.5 mg/mL, or about 2 mg/mL. Sodium azide may be present in the range of 0-0.05%, 0.05-0.03%, or about 0.01% (w/v).
bonding partner

As used herein, "binding partner" refers to any molecule or molecular complex that is capable of specifically binding a target analyte. Binding partners can be, for example, proteins (e.g., antibodies or antigen-binding antibody fragments), small organic molecules, carbohydrates (e.g., polysaccharides), oligonucleotides, polynucleotides, lipids, affinity ligands, aptamers, etc. . In some embodiments, the binding partner is an antibody or fragment thereof. Specific binding in the context of the present invention refers to a binding reaction that determines the presence of a target analyte in the presence of a heterogeneous population. Thus, under certain assay conditions, a given binding partner binds preferentially to a particular protein or isoform of a particular protein and binds in significant amounts to other proteins or other isoforms present in the sample. do not.

In some cases, the antibody is intravenous immunoglobulin (IVIG) and/or an antibody (eg, enriched, purified, eg, affinity purified) from IVIG. IVIG is a blood product that contains IgG (immunoglobulin G) pooled from the plasma of many (e.g., sometimes 1,000 to over 60,000) normal, healthy blood donors (e.g., some (in some cases, without any other proteins). IVIG is commercially available. Aspects of IVIG are described, for example, in U.S. Patent Application Publication Nos. 2010/0150942; 2004/0101909; 2013/0177574; 2013/0108619; , which are hereby incorporated by reference.

When the binding partners are antibodies, they may be monoclonal or polyclonal antibodies. The term "antibody" as used herein refers to immunoglobulin molecules, and immunologically active portions of immunoglobulin (Ig) molecules, that specifically bind to an antigen in a target analyte, for example. Such antibodies include, but are not limited to, polyclonal, monoclonal, monospecific polyclonal antibodies, antibody mimetics, chimeras, single chain, Fab, Fab' and F(ab') ₂ fragments. , Fv, and Fab expression libraries. In some cases, the antibody is a monoclonal antibody of a defined subclass (eg, IgG1, IgG2, IgG3, or IgG4, IgA, IgD, IgE, IgG2a, IgG2b, IgG3, and IgM). When a combination of antibodies is used, the antibodies may be from the same subclass or from different subclasses. For example, the antibody may be an IgG1 antibody. In some embodiments, monoclonal antibodies are humanized. Antibody fragments may include molecules such as Fab, scFv, F(ab')2, and Fab' molecules. Antibody derivatives include added or substituted antibodies or fragments thereof, such as chimeric antibodies. Antibodies may be derived from human or animal sources, from hybridomas, via recombinant methods, or in any other manner known in the art.

Binding partners other than antibodies or antibody fragments or derivatives specific for the target analyte may also be used in the present systems and methods. For example, the binding partner may be a nucleic acid or nucleic acid analog, such as an oligonucleotide or a PNA probe. In one embodiment, aptamers can be used as specific binding partners. Aptamers are single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can bind with high affinity and specificity to preselected targets, including proteins and peptides. Other binding partners capable of binding the target analyte to form receptor-ligand, enzyme-substrate, enzyme-inhibitor, and enzyme-cofactor pairs may also be used. Specific examples of such binding partner pairs include carbohydrate and lectin, biotin and avidin or streptavidin, folic acid and folate binding protein, vitamin B12 and intrinsic factor, protein A and immunoglobulin, and protein G and immunoglobulin. . Also included are binding partners that form covalent bonds with the target analyte.
conjugate

Polymeric dye conjugates may include any known polymeric dye conjugated to a binding partner using techniques known to those skilled in the art. In some embodiments, the polymeric dye is attached to the binding partner using methods for directly modifying the core polymer as described in U.S. Published Application No. 2020/0190253, which is incorporated herein by reference in its entirety. They may also be conjugated to form polymer dye conjugates.

In some cases, the polymeric dye is conjugated to a binding partner using the methods described in U.S. Published Application No. 2019/0144601, which is incorporated herein by reference in its entirety to form a polymeric dye conjugate. may be formed. The method can be illustrated as follows:

SuperNova v428 (SN v428 (Beckman Coulter) is a bright polymer dye that can be activated with an amine for tandem dyes followed by maleimide activation for tandem conjugates. The rigidity of the polymer dye structure is determined by rotation. Helps reduce energy, resulting in brighter luminescence. SuperNova v428 is one of the brightest dyes that can be excited by a violet laser, making it particularly suitable for evaluating dimly expressed markers. SuperNova conjugate The antibodies can include anti-CD19 antibody-SuperNova v428, anti-CD22 antibody-SuperNova v428, anti-CD25 antibody-SuperNova v428, and anti-CD38 antibody-SuperNova v428 antibody-polymer dye conjugate.
target analyte

The present disclosure also provides a method for detecting a target analyte in a sample, wherein the target analyte comprises a target antigen and may be a substance, e.g. a molecule, whose abundance/concentration is determined by several assays. It relates to a method determined by a procedure. The present invention is designed to detect the presence, and in some cases the amount, of a specific target analyte. The term "target analyte" refers to a target molecule, including a target antigen, that is to be detected in a biological sample, such as peptides, proteins, polynucleotides, organic molecules, sugars and other carbohydrates, lipids, etc. as well as small molecules. It is an important aspect of the present disclosure that the target analyte is contained in a liquid sample and is or becomes accessible for binding to a target analyte-specific binding partner of the present invention. It is. Target analytes can be found in biological samples, such as blood samples, cell line development samples, tissue culture samples, and the like.

Target analytes can include, for example, nucleic acids (DNA, RNA, mRNA, tRNA, or rRNA), peptides, polypeptides, proteins, lipids, ions, monosaccharides, oligosaccharides, polysaccharides, lipoproteins, glycoproteins, glycolipids, Or it may be a fragment of these. Target analytes may be proteins, such as structural microfilament, microtubule, and intermediate filament proteins, organelle-specific markers, proteasomes, transmembrane proteins, surface receptors, nuclear pore proteins, protein/peptide trans It may be a locase, a protein folding chaperone, a signaling scaffold, an ion channel, etc. The protein may be an activatable protein or a protein that is differentially expressed or activated in diseased or abnormal cells, including, but not limited to, transcription factors, DNA and/or RNA binding and modification proteins, nuclear including uptake and transport receptors, regulators of apoptosis or survival, and the like.

The target analyte may be present or accessible at the cell surface. Illustrative examples of useful analytes include, but are not limited to: 1) specific cell surface macromolecules and antigens (recognized by hormones, protein complexes, and cell receptors); 2) cellular proteins, DNA or RNA in permeabilized cells that contain abnormal DNA or RNA sequences or abnormal amounts of certain messenger RNAs. Detection of these analytes may be particularly useful in situations where they are found in rare cells and/or as identifiers of rare cells, such as those found in the early stages of various cancers.

In some examples, the target analyte is CD2, CD3, CD4, CD8, CD10, CD11c, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD27, CD38, CD45, CD45RA, CD56, CD62L, CD64 , CD95, CD103, HLA-DR, IFN-γ, TNF-α, or ZAP-70, or other target analytes of interest.
biological sample

Non-limiting examples of biological samples include blood, serum, plasma, urine, semen, breast milk, sputum, mucus, buccal swabs, vaginal swabs, rectal swabs, aspirates, needle biopsies, e.g. , tissue sections obtained by surgery or dissection, plasma, serum, cerebrospinal fluid, lymph, exocrine secretions of the skin, respiratory, intestinal, and genitourinary tracts, lacrimal fluid, saliva, tumors, organs, in vitro cell culture Samples of components include, but are not limited to, conditioned media obtained from cell growth in cell culture media, putative virus-infected cells, recombinant cells, and cellular components.

The sample in the method of the present disclosure may be, for example, blood. The blood sample may be whole blood. Whole blood can be obtained from a subject using standard clinical procedures. The sample comprises one or more cells of whole blood (e.g., red blood cells, white blood cells, lymphocytes (e.g., T cells, B cells, or NK cells), phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, platelets, or any cell with one or more detectable markers). The sample may be from a cell culture. The sample may contain the target analyte naturally or may be prepared in whole or in part through synthetic means.
subject

Subjects may include humans (e.g., patients suffering from or suspected of having the disease), commercially important foods including, for example, cattle, steers, pigs, goats, sheep, birds, fish, or horses. It may also be a chain mammal. Samples may also be obtained from domestic pets or companion animals, including, for example, dogs, cats, rabbits, birds, or ferrets. The subject may be an experimental animal used as an animal model of disease or for drug screening, such as a monkey, mouse, rat, rabbit, or guinea pig. The subject may be an exotic animal, such as a zoo animal or a wild animal, such as an elephant, antelope, zebra, bison, giraffe, lion, tiger, leopard, orangutan, gorilla, whale, dolphin, shark, or reptile. .
reaction vessel

The reaction vessel disclosed herein can be any vessel in which a reaction between a binding partner or its polymer dye conjugate and a target analyte can occur. For example, reaction vessels can be tubes, plates, microtiter plate wells, chambers, and slides. In a preferred embodiment, the reaction vessel has a lid or cap so that the binding reaction can occur in a closed environment.
substrate

The reaction vessel contains one or more substrates. The substrate can be any suitable surface including, but not limited to, plastic, nitrocellulose, cellulose acetate, quartz, and glass. Non-limiting examples of plastics may include polystyrene, polypropylene, cyclo-olefins, and polycarbonates. In some embodiments, the substrate is a membrane. The substrate may be the interior surface of a reaction vessel body, such as a plastic tube or a well of a microtiter plate. The substrate may also be beads. In some embodiments, at least one of the substrates (eg, membranes) that accept labeled binding partners is bound to an interior surface of the reaction vessel body. In some embodiments, the membrane substrate is a sheet or roll, which facilitates solution deposition and facilitates drying. In some embodiments, the membrane may be cut to separate individual dried reaction spots. In some embodiments, the cut membrane is simply dropped into the reaction vessel. In some preferred embodiments, the cut membrane is bonded to the surface of the reaction vessel such that when liquid is pipetted into or out of the reaction vessel, spots do not escape from the vessel.
liquid sample

The reaction vessel is configured to receive a liquid sample. Liquid samples used in the present invention typically contain target analytes obtained as or dispersed in a predominantly aqueous medium.

The sample can be, for example, a biological sample, e.g. blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirate (or any cells obtained from bone marrow), urine (lavage fluid), serum, plasma, saliva, cerebrospinal fluid. fluid, lymph, urine, amniotic fluid, interstitial fluid, stool, mucus, breast milk, semen, buccal swab, nasopharyngeal swab, vaginal swab, rectal swab, aspirate, needle biopsy, e.g., surgical or It may be a tissue section obtained by dissection, or a tissue (eg, tumor sample, non-aggregated tissue, non-aggregated solid tumor) sample. The sample may be a blood sample. The blood sample may be a whole blood sample. Whole blood can be obtained from a subject using standard clinical procedures. The sample may include whole blood (e.g., red blood cells, white blood cells, lymphocytes (e.g., T cells, B cells, or NK cells), phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, (or any cell having one or more detectable markers). Samples include cell cultures, in vitro cell culture components, including, but not limited to, conditioned media obtained from cell growth in cell culture media, presumptive virus-infected cells, recombinant cells, and cell components. It may be from.

The sample may be any source of biological material and may include proteins, carbohydrates, and/or polynucleotides that can be obtained directly or indirectly from living organisms. Samples can include, for example, cells, tissues, or body fluids, as well as deposits left by organisms, including viruses, mycoplasma, and fossils. The sample may contain a target analyte. Target analytes may be naturally occurring in biological samples or may be prepared in whole or in part through synthetic means.
Labeled binding partners

A dye can be conjugated to a binding partner by various coupling chemistries between the reactive pair present in the binding partner and the label. Reactive pairs include, but are not limited to, maleimide/thiol, succimidyl ester (NHS ester)/amine, azide chemistry, carboxy/EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) )/amine, amine/sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol, and amine/BMPH (N-[ ^~ -maleimidopropionic acid]hydrazide.TFA )/thiols are possible. Methods for making conjugations are well known in the art. Commercially available kits for performing conjugations are also available, for example from Innova biosciences (Cambridge, UK), Novus Biologicals (Littleton, Colo.), Thermo Fisher Scientific (Waltham, Mass.). Readily available.

Either dry or liquid polymer dye conjugates can be used in the present methods and compositions. Dry polymer dye conjugates may be prepared using any technique known in the art. The technology may be as described in US2019/0242882, which application is incorporated herein by reference.

Polymer dye conjugates may be used in compositions according to the present disclosure and may be used directly to stain blood and analyze it on a flow cytometer.
assay system

Assay systems for quantifying bound molecules utilizing binding partners and fluorescent labels are well known. Examples of such systems include flow cytometers, scanning cytometers, imaging cytometers, fluorescence microscopes, and confocal fluorescence microscopes.

Flow cytometry is used to detect fluorescence. Several devices suitable for this use are available and known to those skilled in the art. Examples include BCI Navios, Gallios, Aquios, and CytoFLEX™ flow cytometers.

The assay may be an immunoassay. Examples of immunoassays useful in the present invention include, but are not limited to, fluorescence assays (FLA). Assays may also be performed on protein arrays.

Where the binding partner is an antibody, antibody or multiple antibody sandwich assays may also be used. Sandwich assays refer to those that use a series of recognition events to build up layers of different binding partners and report elements that indicate the presence of a particular analyte. Examples of sandwich assays are disclosed in US Pat. No. 4,486,530 and references described therein.

A light source is applied to the sample capable of exciting the polymer and light emitted from the conjugated polymer complex is detected. In a typical assay, fluorescent polymer dye conjugates for use in the present invention are excitable with light having a wavelength between about 395 nm and about 415 nm. The emitted light is typically between about 415 nm and about 475 nm. Alternatively, the excitation light may have a wavelength between about 340 nm and about 370 nm, and the emitted light is between about 390 nm and about 420 nm.
Application

Compositions according to the present disclosure may be monochromatic, ie, include a single polymer dye conjugate, such as a single SN polymer dye conjugate. For example, biological samples may be stained using SN conjugates to monitor or identify specific cell populations depending on the antibody conjugated to the polymer dye.

In some embodiments, compositions according to the present disclosure may include monochromatic polymeric dye conjugates and conventional non-polymeric dye conjugates. For example, SN conjugates can be used with non-polymeric dye conjugates such as CD4-FITC, CD7-PE, CD25-ECD, CD56- It may also be used with PC5.5 etc.

In some embodiments, one or more compositions according to the present disclosure may be contacted with a biological sample, such as a blood sample. For example, a biological sample may be stained with a composition comprising multiple SN conjugates to monitor or identify specific cell populations depending on the antibody conjugated to the polymer dye. In some embodiments, two or more, three or more, or four compositions according to the invention may be contacted with a biological sample. In some embodiments, a composition comprising a plurality of polymeric dye conjugate compositions comprises a non-polymeric dye conjugate, e.g. It may further contain CD4-FITC, CD7-PE, CD25-ECD, CD56-PC5.5, etc.

The invention may be better understood by reference to the following examples, which are provided for illustration. The invention is not limited to the examples given herein.
(Example 1)
Preparation of DHP polymer composite

Method 1: In a round bottom flask, dibromo DHP monomer and diboronic acid DHP monomer (1:1) as described in WO2017/180998 were placed in a (DMF-water) mixture and purged with nitrogen for 10 minutes. Approximately 20 equivalents of CsF and 10% Pd(OAc) were mixed under nitrogen and heated at 80°C. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. A capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture, and after 3 hours a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. Wash using 20% ethanol until absorption of filtrate decreases.

Method 2: Alternatively, polymerization may be carried out by self-polymerizing the bromoboronic ester of the DHP molecule. In a round bottom flask, the DHP bromoboronic ester was placed in a (DMF-water) mixture and purged with nitrogen for 10 minutes. Approximately 10 equivalents of CsF and 5% Pd(OAc) _were mixed under nitrogen and heated at 80 °C. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. A capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture, and after 3 hours a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. Wash using 20% ethanol until absorption of filtrate decreases.

Method 3: In a round bottom flask were placed both dibromodihydrophenanthrene and diboronic acid dihydrophenanthrene monomers (1:1) and THF-water containing 10 equivalents of K ₂ CO ₃ and 3% Pd(PPh ₃ ) ₄ ( 4:1) dissolved in the mixture. The reaction mixture was placed in a Schlenk line, degassed with three freeze-pump-thaw cycles, and then heated to 80° C. with vigorous stirring under nitrogen for 18 hours. A capping agent (selected from G1) containing the appropriate functional group is then added to the reaction mixture via cannula under excess nitrogen pressure, and after 3 hours a second capping agent (selected from G2) is added to the reaction mixture. Added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. Wash using 20% ethanol until absorption of filtrate decreases.

Method 4: Alternatively, polymerization may be carried out by self-polymerizing the bromoboronic acid ester of dihydrophenanthrene molecules. In a round bottom flask, dihydrophenanthrene bromoboronic acid ester was dissolved in a THF-water (4:1) mixture containing 10 equivalents of K ₂ CO ₃ and 3% Pd(PPh ₃ ) ₄ . The reaction mixture was placed in a Schlenk line, degassed with three freeze-pump-thaw cycles, and then heated to 80° C. with vigorous stirring under nitrogen for 18 hours. A capping agent (selected from G1) containing the appropriate functional group is then added to the reaction mixture via cannula under excess nitrogen pressure, and after 3 hours a second capping agent (selected from G2) is added to the reaction mixture. Added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. Wash using 20% ethanol until absorption of filtrate decreases.
(Example 2)
Preparation of fluorene-DHP copolymer composite

Method 1: In a round bottom flask, both dibromoDHP and diboronic acid fluorene monomer (1:1) were placed in a (DMF-water) mixture and purged with nitrogen for 10 minutes. Approximately 20 equivalents of CsF and 10% Pd(OAc) were mixed under nitrogen and heated at 80°C. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. A capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture, and after 3 hours a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. Wash using 20% ethanol until absorption of filtrate decreases.

Method 2: In a round bottom flask, both dibromofluorene and diboronic acid DHP monomer (1:1) were placed in a (DMF-water) mixture and purged with nitrogen for 10 minutes. Approximately 20 equivalents of CsF and 10% Pd(OAc) were mixed under nitrogen and heated at 80°C. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. A capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture, and after 3 hours a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. Wash using 20% ethanol until absorption of filtrate decreases.

Method 3: In a round bottom flask, place both dibromodihydrophenanthrene and diboronic acid fluorene monomers (1:1) and THF-water containing 10 equivalents of K ₂ CO ₃ and 3% Pd(PPh ₃ ) ₄ ( 4:1) dissolved in the mixture. The reaction mixture was placed in a Schlenk line, degassed with three freeze-pump-thaw cycles, and then heated to 80° C. with vigorous stirring under nitrogen for 18 hours. A capping agent (selected from G1) containing the appropriate functional group is then added to the reaction mixture via cannula under excess nitrogen pressure, and after 3 hours a second capping agent (selected from G2) is added to the reaction mixture. Added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. Wash using 20% ethanol until absorption of filtrate decreases.

Method 4: In a round bottom flask, place dibromofluorene and diboronic acid dihydrophenanthrene monomers (1:1) and add THF-water (4:1 ₎ containing 10 equivalents of _K2CO3 and 3% Pd(PPh3)4. ) dissolved in the mixture. The reaction mixture was placed in a Schlenk line, degassed with three freeze-pump-thaw cycles, and then heated to 80° C. with vigorous stirring under nitrogen for 18 hours. A capping agent (selected from G1) containing the appropriate functional group is then added to the reaction mixture via cannula under excess nitrogen pressure, and after 3 hours a second capping agent (selected from G2) is added to the reaction mixture. Added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. Wash using 20% ethanol until absorption of filtrate decreases.
(Example 3)
Comparison of fluorescence emission spectra

A comparison of the fluorescence emission spectra of fluorene (Fl-Fl), dihydrophenanthrene (DHP-DHP) and fluorene-DHP (DHP-Fl) polymers was performed. After excitation at 405 nm, the DHP-containing polymer shows a significant difference in its fluorescence maximum wavelength between 426 and 428 nm, while the fluorene-based polymer exhibits a maximum wavelength of 421 nm, as shown in FIG. 1A.
(Example 4)
Comparison of absorption spectra

Absorption spectra of both fluorene (Fl-Fl) and dihydrophenanthrene (DHP-DHP) polymers were measured. The DHP-DHP polymer (black curve) exhibits a lambda max (λmax) at 390 and 410 nm, while the Fl-Fl (gray curve) polymer exhibits a lambda max (λmax) around 400 nm, as shown in Figure 1B. . Samples were measured under different concentrations.
(Example 5)
Polymer dye properties

The polymeric dyes of the present disclosure have been found to have certain physical and chemical properties, such as absorption, fluorescence, brightness, molecular weight, polydispersity, and dye to protein ratio when conjugated to an antibody. . Preferred ranges for these parameters are shown in Table 1A.

The excitation and emission spectra of the tandem polymer were measured. Excitation was performed at the polymer maximum wavelength (405 nm) and emission was observed from various acceptor dyes attached to the backbone.
(Example 6)
Experiments with unconjugated dyes

Blood samples were stained with non-conjugated polymer dye SN v605 with Empigen BB® (no antibody) and non-conjugated polymer dye SN v605 without Empigen BB® (no antibody) and analyzed with a flow cytometer. As shown in the bottom right of Figure 2, the presence of EMPIGEN BB® shows an effective reduction in the non-specific interaction of the fluorescent polymer conjugated to the binding partner to leukocytes in the blood. Ta. In Figure 2, blood samples were stained using the antibody-free fluorescent polymer dye SN v605 and analyzed on a flow cytometer. At the bottom left of Figure 2, it is evident that the polymeric dye without Empigen BB® binds non-specifically to monocytes/granulocytes. Without wishing to be bound by any particular theory, it appears likely that the polymer is adsorbed to the cell surface of monocytes and granulocytes. When EMPIGEN BB® is added, the cell surface is blocked by EMPIGEN BB® molecules and non-specific binding of the polymer to monocytes and granulocytes is substantially reduced.

With EMPIGEN BB® added to the polymer, the dot plot appears similar to that of the unstained sample in the top panel of Figure 2 without any polymer. We show that the spread of the granulocyte population corresponds to unstained tubes and that when EMPIGEN BB® is added to the polymeric dye, the nonspecific interaction of the polymeric dye with monocytes and granulocytes is dramatically reduced. suggest.
(Example 7)
Experiments with conjugated dye SN 605-CD20

EMPIGEN BB® can be combined with the conjugates described herein (e.g., SN605-CD20, SN786-CD103, and SN428 conjugates), bovine serum albumin (BSA; 2 mg/mL), sodium azide (0.5 mg/mL), 1%), and pluronic F-68 (polyethylene oxide-polypropylene oxide-polyethylene oxide nonionic triblock copolymer) at a dose of 0.12% per 10 μl of conjugate.

CD20 is a B-lineage cell marker that is expressed during pre-B lymphocyte development, persists in B lymphocyte expression, and loses its expression upon plasma cell differentiation. CD20 is not expressed on other leukocyte populations including monocytes, granulocytes, and NK cells. FIG. 3 shows the performance of SN 605-CD20 conjugates without and in the presence of EMPIGEN BB®.

The percentage of non-specifically bound granulocytes decreased with the use of EMPIGEN BB® (see "P2" gate in the dot plot). The functional aspects of the conjugate were also unchanged as the percentage of positive population was similar in both cases (see "P1" gate in the dot plot).

To confirm the effectiveness of EMPIGEN BB®, two lots of SN 605-CD20 conjugate were tested in the presence and absence of EMPIGEN BB®. The mean fluorescence intensity (MFI) was compared to the autofluorescence of monocytes from unstained samples (median fluorescence intensity of the negative population (MdFI)). The results shown in Figure 4 demonstrate that in the presence of detergent, nonspecific interactions on monocytes were reduced to 75% and 67% for Lot 1 and Lot 2 of the SN605 CD20 conjugate, respectively. There is. A similar effect (reduction in non-specific binding) was also observed in granulocytes, although the percentage reduction was less pronounced (13.7% and 17.7% for Lot 1 and Lot 2, respectively, Figure 5).
(Example 8)
Experiments with conjugated dye SN 786-CD103

Since the CD103 conjugate is not normally expressed in normal whole blood, its testing is performed in a cell line (MOLT16). Since there is no expression of CD103 in normal blood, there should be no positive signal. However, due to non-specific interactions, the SN786 CD103 conjugate also tends to bind whole blood. Adding EMPIGEN BB® to this formulation helps contain non-specific interactions. This is shown in FIG.

Two lots of SN 786-CD103 conjugate were tested in the presence and absence of EMPIGEN BB® to compare autofluorescence of monocyte and granulocyte populations (MdFI of negative population) to unstained samples. do. As shown in Figure 7, the signal from non-specific binding of the conjugate to monocytes was reduced by 149.3% and 202.1% for Lot 1 and Lot 2 of SUPERNOVA™ conjugate, respectively. The same effect (reduced non-specific binding) was similarly observed for the granulocyte population, with a 150.8% and 253% reduction for Lot 1 and Lot 2, respectively, as shown in Figure 8.
(Example 9)
Effect of zwitterionic surfactants on monocyte background reduction using SN 428 conjugate

The efficiency of the zwitterionic surfactant EMPIGEN BB® in reducing non-specific binding to monocytes was evaluated. Other populations, namely lymphocytes and granulocytes, were also studied to assess the no effect of detergent on MFI and cell percentage.

Experimental conditions were generally as follows:
- CD19-SN 428 (Lot D19-094, Polymer Lot RDS-042919 (82.7 kD), 1 dose (0.5 μg/test).
- CD22-SN 428 (Lot D19-109, Polymer Lot WX-20190624 (86.4 kD), 1 dose (0.5 μg/test).
- CD25-SN 428 (Lot D19-107, Polymer Lot RDS-062419 (72.8 kD), 1 dose (0.5 μg/test).
- Becton Dickinson CD19, CD22 and CD25-BV 421, commercially available dose 1×.
- Three doses of EMPIGEN BB® were prepared as conjugate final formulations: 0.06%, 0.12%, and 0.2%.
- Added 10 μL of sample and 100 μL of whole blood.
- Two donors were tested and lysed using VersaLyse (a lysis solution used to lyse red blood cells, Beckman Coulter, Inc.) + Fix, one wash.
- Obtained Navios flow cytometer at FL9.

The required number x of tubes was prepared, the "number x of tubes" depending on the performance being tested. The calculated volume of conjugated antibody (at the required dose) was added to each tube. Whole blood (100 μL) was added into each tube. The tubes were gently vortexed for 15 seconds and incubated for 15 to 20 minutes at room temperature, 18-25°C, protected from light. VersaLyse and IOTest3 fixative mixture (2 mL of Versalyse Ref. A09777 + 10 x 50 μl of IOTest3 fixative Ref. A07800) was added to the tube. The tubes were immediately vortexed for 1 second and incubated for 10 minutes at room temperature (18-25°C), protected from light. The tubes were centrifuged at 300 g for 5 min at room temperature, the supernatant was removed by aspiration, and the cell pellet was resuspended using 1× 3 mL of PBS. The tubes were centrifuged again at 300 g for 5 min at room temperature (18-25°C), the supernatant was removed by aspiration, and the cell pellet was resuspended using 0.5 ml of PBS 1X or PBS 1X formaldehyde 0.1%. suspended (can be obtained by diluting 1× 1 ml of PBS + 12.5 μl of IOTest3 fixative 10×).

In cytometry, correction is the mathematical correction of signal overlap between channels of the emission spectra of different fluorochromes. Therefore, this modified count was used to exclude signal leakage into other undesired channels. Manual corrections were performed to evaluate conjugate performance.

Raw data obtained on two donors using CD19, CD22 and CD25 SUPERNOVA® v428 conjugate, data normalized to conditions without EMPIGEN®, and final monocyte background staining. The % reduction is shown in Table 1B. The data show that in the presence of Empigen BB®, non-specific background monocyte binding is between 52 and 73% compared to conditions without EMPIGEN®, where monocyte background is maximal. This indicates a substantial decline in

Data showing the effect of Empigen BB® on granulocyte background reduction is shown in Table 2. Granulocyte background reductions were observed ranging from 4 to 21% compared to conditions without EMPIGEN® where granulocyte background was maximal.

Table 3 shows the effect of EMPIGEN® on the positive lymphocyte population: the presence of EMPIGEN® significantly alters the positive signal for lymphocytes when compared to conditions without EMPIGEN®. did not induce.

The data in Tables 1-3 are summarized in Figures 11-15.

Table 4 describes additional experiments and shows the percent reduction relative to background monocytes and granulocytes.

(Example 10)
Effect of EMPIGEN BB® on sample cell integrity

EMPIGEN BB® is a detergent and therefore may result in permeabilization of cell membranes and cause cell death. From the studies described herein, the concentration of EMPIGEN BB® used with the polymer dye conjugate does not induce permeabilization or killing of whole blood cells and does not affect the performance of the conjugate. It was concluded.

Micelle concentration studies were performed in the samples and during the staining period to ensure that the critical micelle concentration (CMC) was not exceeded. Referring to Table 5, it shows the evaluation of CMC in the conjugate formulation and during the staining period. CMC studies were performed in the conjugate formulation and during the staining period in 100 μL of whole blood. Experiments were conducted to evaluate the effect of addition of EMPIGEN BB® on whole blood cell integrity and on peripheral blood mononuclear cells (PMBC).

The percentage of dead cells in whole blood samples containing 7-AAD was evaluated. 7-AAD is a DNA marker, and stains positive when the cell membrane becomes permeable. CD19-SNv428 D19-094 without EMPIGEN BB(R) (negative control) and with 0.06%, 0.12%, and 0.2% EMPIGEN BB(R), 4 people including 7-ADD The test was performed on whole blood from 100 donors to assess the percentage of dead cells in each condition. The goal of the experiment was to establish whether the percentage of dead cells was increased by EMPIGEN BB® concentration.

Two whole blood samples stored for more than 24 hours were included as positive controls for 7-AAD staining.

7-AAD staining protocol:
- 100 μl of whole blood + 10 μl of CD19-SNv428+/- Empigen, incubate for 20 minutes.
・Dissolve using Versalyse, wash once,
- Resuspend in 500 μl 1x PBS - Add 20 μl of 7-AAD (Ref B88526) and incubate for 15-20 minutes - Navios flow cytometer acquisition: FL4 for 7-AAD, FL9 for CD19-SNv428

The data are shown in FIG.

Finally, EMPIGEN BB(R) has been shown to be effective in reducing non-specific interactions of the polymer dye conjugates described herein (SN 605-CD20, SN786-CD103, SN428-CD25, SN428-CD19 and SN428-CD22). It has been proven to be effective in reducing EMPIGEN BB® efficiently reduces non-specific background binding with monocyte and granulocyte populations when tested against the conjugate's five specificities. This efficiency of EMPIGEN BB® is donor independent. When the conjugate was compared to BV786-CD103 and BV 421, a clear difference in reduction of monocyte non-specific extraction was observed. In addition to its performance, the presence of EMPIGEN BB® and the polymer dye conjugate does not induce whole blood cell membrane permeabilization and inhibits whole blood cell death at concentrations up to at least 0.2% in the composition. I didn't induce it.
(Example 11)
Effects of nonionic surfactants

The nonionic surfactants Tween®-20, tergitol, NP-40, and Pluronic F-68 (PF-68) inhibit the nonspecific binding of the conjugates described herein to monocytes. Additional detergents/surfactants were tested for removal. Figure 9 shows the inefficiency of Tween®-20 and PF-68 in avoiding non-specific binding of the conjugate to monocytes.
(Example 12)
Effect of protein blockers

The problem of non-specific interaction with monocytes was also observed with conventional tandem dyes (eg, PC5, PC5.5, PC7, AA700, available from Beckman Coulter, Inc.). To overcome this problem, we considered that the known protein blockers BSA-ox (oxidized BSA) and BSA-Cy5-ox (oxidized Cy5-BSA) could block non-specific binding. However, it was observed that BSA, BSA-ox, and BSA-Cy5-ox were all inefficient in controlling nonspecific interactions of polymer dye conjugates with granulocytes/monocytes. . Please refer to FIG.
(Example 13)
Effect of anionic surfactants on nonspecific binding

The anionic surfactant N-laurylsarcosine (NLS) was shown to be effective at reducing nonspecific staining on monocytes and granulocytes at 0.16% and 0.08% (w/v). Admitted.

NLS is an anionic surfactant with a CMC of 14.57mM (30°C). NLS sodium was evaluated to determine the effective concentration to block non-specific binding of the polymer dye conjugate (SN v605-CD20) to monocytes and granulocytes.

In this example, different concentrations (0.16%, 0.08%, 0.04% and 0.02% w/v) of NLS were tested using SN v605CD20 conjugate in the presence of BSA and sodium azide. Formulated and stained peripheral blood samples. Flow cytometry was performed after sample staining.

Figures 17A-E show point-plots of blood samples in the absence and presence of different concentrations of conjugate with/without Empigen/NLS. A dot plot of a single color conjugate-free peripheral blood sample is shown in Figure 17A, and it is clear that no population is observed in the CD20+ gate.

Positive control point plot of a peripheral blood sample in the presence of CD20-SN v605 monochromatic conjugate in a buffer composition containing BSA, sodium azide, and the zwitterionic surfactant Empigen BB® as additives. is shown in FIG. 17B. When compared to the negative control point plot (Figure 17C), the population % in the gates "monocyte non-specific binding" (0.64%) and "granulocyte non-specific binding" (0.68%) were each significantly 2, demonstrating the effectiveness of Empigen BB® in blocking non-specific binding to monocytes and granulocytes.

A negative control point plot of a peripheral blood sample in the presence of a CD20-SN v605 monochromatic conjugate in a buffer composition containing only BSA and sodium azide as additives is shown in FIG. 17C. Without detergent, granulocyte non-specific staining was 1.20% and monocyte non-specific staining was 1.63%.

A test point plot of a peripheral blood sample in the presence of CD20-SN v605 monochromatic conjugate in a buffer composition containing BSA, sodium azide, and NLS (0.16% w/v) as additives is shown in Figure 17D. show. Granulocyte non-specific staining was substantially reduced to 0.60% and monocyte non-specific staining was substantially reduced to 0.39% compared to the negative control (FIG. 17C).

A test point plot of a peripheral blood sample in the presence of CD20-SN v605 monochromatic conjugate in a buffer composition containing BSA, sodium azide, and NLS (0.08% w/v) as additives is shown in Figure 17E. show. Compared to the negative control (Figure 17C), granulocyte non-specific staining was reduced to 0.76% and monocyte non-specific staining was reduced to 0.93%.

The point plot in Figure 17B (Empigen BB®) shows that the population % in the gates for "monocyte non-specific binding" and "granulocyte non-specific binding" when compared to Figures 17D and 17E (NLS). The efficacy of NLS equivalent to Empigen in blocking non-specific binding to cells is shown to be 0.16% and 0.08%.

In CD20-SN v605, the effective concentration of NLS to reduce or eliminate non-specific staining for monocytes and granulocytes was found to be 0.16% to 0.08% w/v, thus This concentration range of NLS anionic surfactants was demonstrated to be effective in reducing non-specific binding in monochromatic fluorescent polymer dye conjugate compositions.

Claims (31)

A method for reducing or eliminating non-specific binding of at least one polymer dye conjugate in a biological sample, the method comprising:
contacting the at least one polymeric dye conjugate with at least one zwitterionic or anionic surfactant before, during, or after contacting the polymeric dye conjugate with a blood sample; wherein said contacting results in reduced non-specific binding of said at least one polymeric dye conjugate to cells in said biological sample. 2. The method of claim 1, wherein said reducing non-specific binding comprises reducing non-specific binding to leukocytes in said biological sample; optionally, said biological sample is a blood sample. . 3. The method of claim 2, wherein the leukocytes are selected from the group consisting of monocytes and granulocytes. 4. The method of any one of claims 1 to 3, comprising contacting the biological sample with the surfactant before contacting the polymer dye conjugate with the biological sample. 4. Any one of claims 1 to 3, comprising contacting the polymer dye conjugate and the surfactant before contacting the polymer dye conjugate and the surfactant with the biological sample. The method described in. The surfactant has the formula:
R ^1' [CO-X(CH ₂ ) _j ] _g - [N ⁺ (R ^2' ) (R ^3' )] _k - (CH ₂ ) _f - [CH(OH)CH ₂ ] _h -Y ^-
(In the formula,
R ^1′ is saturated or unsaturated C _5-24 alkyl;
X is NH, NR ⁴ ′, where R ^4′ is C _1-4 alkyl, O, or S;
j is an integer from 1 to 10;
g is 0 or 1;
R ^2' and R ^3' are independently C _1-4 alkyl;
k is 0 or 1;
Hydroxyl is optionally substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl;
f is an integer from 0 to 4;
h is 0 or 1;
Y is COO, SO ₃ , OPO(OR ^5' )O, or P(O)(OR ^5' )O, where R ^5' is H or C _1-4 alkyl)
2. The method according to claim 1, wherein the compound is a compound of The surfactant has the formula:
R ^1' [CO-X(CH ₂ ) _j ] _g -N ⁺ (R ^2' )(R ^3' )-(CH ₂ ) _f - [CH(OH)CH ₂ ] _h -Y ^-
(In the formula,
R ^1′ is saturated or unsaturated C _5-24 alkyl;
X is NH or NR ^4' , where R ^4' is C _1-4 alkyl, O, or S;
j is an integer from 1 to 10;
g is 0 or 1;
R ^2' and R ^3' are independently C _1-4 alkyl;
Hydroxyl is optionally substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl;
f is an integer from 1 to 4;
h is 0 or 1;
Y is COO, SO ₃ , OPO(OR ^5' )O, or P(O)(OR ^5' )O, where R ^5' is H or a C _1-4 alkyl residue)
7. A method according to any one of claims 1 to 6, wherein the zwitterionic surfactant compound is a zwitterionic surfactant compound. The zwitterionic surfactant has the formula:
R ^1' -N ⁺ (CH ₃ ) ₂ -CH ₂ COO ^- ;
R ^1' -CO-NH(CH ₂ ) ₃ -N ⁺ (CH ₃ ) ₂ -CH ₂ COO ^- ;
R ^1' -N ⁺ (CH ₃ ) ₂ -CH ₂ CH(OH)CH ₂ SO ₃ ^- ; or R ^1' -CO-NH-(CH ₂ ) ₃ -N ⁺ (CH ₃ ) ₂ -CH ₂ CH (OH)CH ₂ SO ₃ ^-
8. The method according to claim 7, wherein the compound is a compound of The surfactant is almond amidopropyl betaine, apricot amidopropyl betaine, avocado amidopropyl betaine, babasamidopropyl betaine, behenamidopropyl betaine, behenyl betaine, canolamidopropyl betaine, caprylic/capramidopropyl betaine, carnitine, Cetyl betaine, cocamidoethyl betaine, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, coco betaine, coco hydroxysultaine, coco/oleamidopropyl betaine, coco sultaine, decyl betaine, dihydroxyethyl oleyl glycinate, dihydroxyethyl soy glycinate , dihydroxyethyl stearyl glycinate, dihydroxyethyl tallow glycinate, dimethicone propyl PG-betaine, dolcamidopropyl hydroxysultaine, hydrogenated tallow betaine, isostearamidopropyl betaine, lauramide propyl betaine, lauryl betaine, lauryl hydroxysultaine, Lauryl Sultaine, Milkamide Propyl Betaine, Milkamide Propyl Betaine, Myristamide Propyl Betaine, Myristyl Betaine, Oleamide Propyl Betaine, Oleamide Propyl Hydroxy Sultaine, Oleyl Betaine, Olive Amidopropyl Betaine, Palmamide Propyl Betaine, Palmitamide Propyl betaine, palmitoyl carnitine, palm kernel amidopropyl betaine, polytetrafluoroethylene acetoxypropyl betaine, ricinolamide propyl betaine, sesamidopropyl betaine, soy amidopropyl betaine, stearamide propyl betaine, stearyl betaine, tallow amidopropyl betaine, tallow amide 9. The method of any one of claims 1 to 8, selected from the group consisting of propylhydroxysultaine, tallow betaine, tallow dihydroxyethylbetaine, undecylenamidopropylbetaine, and wheat germ amidopropylbetaine. 10. A method according to any one of claims 1 to 9, wherein the surfactant is lauryl betaine. The surfactant has the formula:
R ^1' [CO-X(CH ₂ ) _j ] _g -(CH ₂ ) _f -[CH(OH)CH ₂ ] _h -Y ^- (in the formula,
R ^1′ is saturated or unsaturated C _5-24 alkyl;
X is NH, NR ⁴ ′, where R ^4′ is C _1-4 alkyl, O, or S;
j is an integer from 1 to 10;
g is 0 or 1;
Hydroxyl is optionally substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl;
f is an integer from 0 to 4;
h is 0 or 1;
Y is COO, SO ₃ , OPO(OR ^5' )O or P(O)(OR ^5' )O, where R ^5' is H or C _1-4 alkyl)
anionic surfactant compound according to any one of claims 1 to 6, wherein the anionic surfactant may be in acidic form or in the form of its sodium or potassium salt. the method of. The anionic surfactant has the formula R ^1' -CO-N(CH ₃ )-CH ₂ -COO ^- ; or R ^1' -CO-N(CH ₃ )-CH ₂ -SO ₃ -
(In the formula,
R ^1' is saturated or unsaturated C _5-24 alkyl)
12. The method of claim 11, wherein the anionic surfactant is an acidic form or a sodium or potassium salt thereof. The anionic surfactant is N-lauroylsarcosine, sodium lauroylsarcosinate, sodium palmitoylsarcosinate, sodium stearoylsarcosinate, N-methyl-N-(1-oxotetradecyl)-glycine sodium salt, caproylsarcosinate. From the group consisting of sodium, sodium capryloyl sarcosinate, N-methyl-N-(1-oxo-9-octadecen-1-yl)-glycine, sodium salt, sodium oleoyl sarcosinate, and sodium linoleoyl sarcosinate 13. The method according to claim 11 or 12, wherein the method is selected. 14. The method according to claim 13, wherein the anionic surfactant is N-lauroylsarcosine or a salt thereof. The polymer dye conjugate has formula III:
(In the formula,
each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers;
Each optional M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap modifying monomers, optionally substituted ethylene, and ethynylene and is uniform along the polymer backbone. or randomly distributed;
Each optional L is a linker part;
each G ¹ and G ² is independently selected from an unmodified polymer terminus and a modified polymer terminus;
a, c, and d define the mol% of each unit, each of which may be repeated uniformly or randomly, each a being a mol% from 10 to 100%, and each c being a mol% of 10 to 100%. , 0 to 90% mol%, and each d is 0 to 25% mol%;
each b is independently 0 or 1;
each m is an integer from 1 to about 10,000)
15. A method according to claims 1 to 14, comprising a binding partner conjugated to a polymeric dye having the structure. 16. The method of claim 15, wherein A is selected from the group consisting of a DHP moiety, a fluorene moiety, and a DHP moiety and a fluorene moiety. The at least one polymeric dye conjugate has formula I:
(In the formula,
each X is independently C or Si;
each Y is independently CR ¹ R ² or SiR ¹ R ² ;
Each R ¹ is independently an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a moiety:
And;
Each R ² is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, Ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, or moieties
And;
each R ³ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups;
each Z is independently selected from the group consisting of C, O, and N;
each Q is independently selected from the group consisting of a bond, NH, ^NR4 , and _CH2 ;
Each subscript n is independently an integer from 0 to 20;
each M is a unit capable of modifying the polymer bandgap and is uniformly or randomly distributed along the polymer backbone;
L is a linker;
G ¹ and G ² are hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate , phosphates, boronic acid substituted aryls, boronic ester substituted aryls, boronic esters, boronic acids, optionally substituted dihydrophenanthrenes (DHP), optionally substituted fluorenes; amines, carbamates, carboxylic acids, one or more functional groups terminated with carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazine, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protecting groups thereof. each independently selected from the group consisting of aryl or heteroaryl substituted with a pendant chain;
a, c, and d each define the mol% of each unit in the structure, which may be uniformly or randomly repeated; a is mol% from 10 to 100%; c is mol% from 0 to 90%, and each d is mol% from 0 to 25%;
each b is independently 0 or 1;
m is an integer from 1 to about 10,000;
each n is independently an integer from 1 to 20)
17. A method according to any one of claims 1 to 16, comprising a binding partner conjugated to a polymer having a structure according to the invention. L is uniformly or randomly distributed along the polymer backbone; amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone; , azides, alkynes, aldehydes, thiols, and their protecting groups conjugated to the binding partner, substituted with one or more pendant chains terminating in an aryl or heteroaryl group. 18. The method according to claim 17. 19. A method according to any one of claims 15 to 18, wherein the binding partner is a molecule or molecular complex capable of specifically binding a target analyte. 20. A method according to any one of claims 15 to 19, wherein the binding partner is a protein, an affinity ligand, an antibody, or an antibody fragment. The group wherein said binding partner consists of monoclonal antibodies, polyclonal antibodies, immunoglobulins, immunologically active portions of immunoglobulins, single chain antibodies, Fab fragments, Fab' fragments, and F(ab')2 fragments, and scFv fragments. 21. The method of claim 20, wherein the method is selected from: Polymer dye conjugates;
A composition comprising an aqueous buffer; and a zwitterionic or anionic surfactant. 23. The composition of claim 22, further comprising a non-polymeric dye conjugate. 24. A composition according to claim 22 or 23, wherein the concentration of the zwitterionic or anionic surfactant is below the critical micelle concentration (CMC). The concentration of the surfactant is 0.05 to 0.25% (w/v), 0.06 to 0.20% (w/v), or 0.08 to 0.16% (w/v) 25. The composition of claim 24. 26. According to any one of claims 22 to 25, the aqueous buffer comprises one or more additional additives selected from the group consisting of protein stabilizers, preservatives, and additional surfactants. Compositions as described. After contacting said composition with a biological sample, said polymeric dye conjugate causes said polymeric dye conjugate to contact said sample without a zwitterionic or anionic surfactant. 27. The composition of any one of claims 22 to 26, wherein the composition exhibits reduced non-specific binding to cells in the sample compared to non-specific binding of the polymer dye conjugate to cells in the sample. 28. The composition of claim 27, wherein the sample is a blood sample and the cells are leukocytes selected from the group consisting of monocytes and granulocytes. The surfactant has the formula:
R ^1' [CO-X(CH ₂ ) _j ] _g - [N ⁺ (R ^2' ) (R ^3' )] _k - (CH ₂ ) _f - [CH(OH)CH ₂ ] _h -Y ^- ( During the ceremony,
R ^1′ is saturated or unsaturated C _5-24 alkyl;
X is NH, NR ⁴ ′, where R ^4′ is C _1-4 alkyl, O, or S;
j is an integer from 1 to 10;
g is 0 or 1;
R ^2' and R ^3' are independently C _1-4 alkyl;
k is 0 or 1;
Hydroxyl is optionally substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl;
f is an integer from 0 to 4;
h is 0 or 1;
Y is COO, SO ₃ , OPO(OR ^5' )O, or P(O)(OR ^5' )O, where R ^5' is H or C _1-4 alkyl)
29. A composition according to any one of claims 22 to 28, wherein when k=0, the surfactant may be in acidic form or its sodium or potassium salt. . The polymer dye conjugate has formula III:
(In the formula,
each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers;
Each optional M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap modifying monomers, optionally substituted ethylene, and ethynylene and is uniform along the polymer backbone. or randomly distributed;
Each optional L is a linker part;
each G ¹ and G ² is independently selected from an unmodified polymer terminus and a modified polymer terminus;
a, c, and d define the mol% of each unit, each of which may be repeated uniformly or randomly, each a being a mol% from 10 to 100%, and each c being a mol% of 10 to 100%. , 0 to 90% mol%, and each d is 0 to 25% mol%;
each b is independently 0 or 1;
each m is an integer from 1 to about 10,000)
30. A composition according to any one of claims 22 to 29, comprising a binding partner conjugated to a polymeric dye having the structure. The polymer dye conjugate has formula I:
(In the formula,
each X is independently C or Si;
each Y is independently CR ¹ R ² or SiR ¹ R ² ;
Each R ¹ is independently an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a moiety:
And;
Each R ² is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, Ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, or moiety:
And;
each R ³ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups;
each Z is independently selected from the group consisting of C, O, and N;
each Q is independently selected from the group consisting of a bond, NH, ^NR4 , and _CH2 ;
Each subscript n is independently an integer from 0 to 20;
each M is a unit capable of modifying the polymer bandgap and is uniformly or randomly distributed along the polymer backbone;
L is a linker;
G ¹ and G ² are hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate , phosphates, boronic acid substituted aryls, boronic ester substituted aryls, boronic esters, boronic acids, optionally substituted dihydrophenanthrenes (DHP), optionally substituted fluorenes; amines, carbamates, carboxylic acids, one or more functional groups terminated with carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazine, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protecting groups thereof. each independently selected from the group consisting of aryl or heteroaryl substituted with a pendant chain;
a, c, and d each define the mol% of each unit in the structure, which may be uniformly or randomly repeated; a is mol% from 10 to 100%; c is mol% from 0 to 90%, and each d is mol% from 0 to 25%;
each b is independently 0 or 1;
m is an integer from 1 to about 10,000;
each n is independently an integer from 1 to 20)
31. A composition according to any one of claims 22 to 30, comprising a binding partner conjugated to a polymeric dye having a structure according to .