CN116997797A

CN116997797A - Additives for reducing non-specific interactions between fluorescent polymer conjugates and cells in biological samples

Info

Publication number: CN116997797A
Application number: CN202180088861.8A
Authority: CN
Inventors: 拉杰什·文卡特什; 希瓦·兰吉尼·斯里尼瓦桑; 瓦莱丽·马利特德西涅; 西尔万·蒙索奥; 杰拉尔丁·比歇勒
Original assignee: Coulter International Corp
Current assignee: Beckman Coulter Inc
Priority date: 2020-11-13
Filing date: 2021-11-12
Publication date: 2023-11-03
Also published as: AU2021380843A1; CA3198558A1; WO2022104147A1; JP2023550721A; EP4244625A1

Abstract

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

Description

Additives for reducing non-specific interactions between fluorescent polymer conjugates and cells in biological samples

The present application was filed as PCT international patent application at 2021, 11 and 12, and claims priority from U.S. provisional application serial No. 63/113,703 filed at 2020, 11 and 13, which is incorporated herein by reference in its entirety.

Background

The polymer dye conjugate (conjugate) is bright and provides excellent properties useful in single or multi-color flow cytometry assays. In general, polymer dye conjugates exhibit high brightness due to their unique and complex structure. But the same unique and complex structure may also lead to some significant limitations. The present disclosure addresses these limitations.

Disclosure of Invention

Due to the nature of the polymer dye conjugate, it can bind non-specifically to cells in biological samples (e.g., monocytes and granulocytes in peripheral blood samples). Nonspecific binding can lead to misinterpretation, leading to false positive inferences. For example, during analysis of cell markers, when the polymeric dye conjugate is contacted with blood, the conjugate may bind non-specifically to cells (e.g., monocytes and/or granulocytes), thereby giving a signal that may be misinterpreted as a positive population or pulling out (pull out) a population that is not desired.

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 compositions for reducing or eliminating non-specific binding of a dye conjugate to a cell in a biological sample, the compositions comprising a dye conjugate as described herein and a surfactant.

In some embodiments, the present disclosure provides methods for reducing or eliminating non-specific binding of at least one dye conjugate to a cell in a biological sample, the methods comprising: contacting at least one dye conjugate with at least one zwitterionic surfactant prior to, during or after contacting the dye conjugate with the biological sample, the contacting resulting in reduced non-specific binding of the at least one dye conjugate in the sample. In some embodiments, the present disclosure provides methods for reducing or eliminating at least one non-specific binding to cells in a blood sample, the methods comprising: contacting the at least one dye conjugate with at least one anionic surfactant prior to, during or after contacting the dye conjugate with the blood sample, the contacting resulting in reduced non-specific binding of the at least one dye conjugate in the blood sample. 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.

Methods for reducing or eliminating non-specific binding of at least one dye conjugate in a biological sample (e.g., a blood sample) are provided, the methods comprising: contacting the at least one dye conjugate with at least one zwitterionic or anionic surfactant prior to, during or after contacting the polymer dye conjugate with the biological sample, the contacting resulting in reduced non-specific binding of the at least one polymer dye conjugate in the biological sample.

In some embodiments, the biological sample may be a blood sample. In some embodiments, the cells may be leukocytes, and the reduced non-specific binding may comprise reduced non-specific binding to leukocytes in the blood sample. In some embodiments, the leukocytes are selected from monocytes and granulocytes.

In some embodiments, the method comprises adding the surfactant to the polymer dye conjugate prior to contacting the polymer dye conjugate with the biological sample (e.g., a peripheral blood sample).

In some embodiments, the method comprises adding the surfactant to the blood sample prior to contact with the polymer dye conjugate.

The surfactant may be a compound of 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 ^- wherein

R ^1’ Is saturated or unsaturated C _5-24 An alkyl group;

x is NH, NR ^4’ Wherein R is ^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 is ^3’ Independently C _1-4 An alkyl group;

k is 0 or 1;

hydroxy is optionally substituted with methyl, ethyl, hydroxymethyl or hydroxyethyl;

f is an integer from 0 to 4;

h is 0 or 1; and is also provided with

Y is COO, SO ₃ 、OPO(OR ^5' ) O OR P (O) (OR) ^5' ) O, where R is ^5’ Is H or C _1-4 Alkyl, and when k=0, the surfactant may be in an acidic form or a sodium or potassium salt thereof.

In some embodiments, the surfactant may be a zwitterionic surfactant compound of the formula:

R ^1′ [CO-X(CH ₂ ) _j ] _g -N ⁺ (R ^2’ )(R ^3′ )-(CH ₂ ) _f -[CH(OH)CH ₂ ] _h -Y ^- ，

wherein:

R ^1’ is saturated or unsaturated C _5-24 An alkyl group;

x is NH or NR ^4’ Wherein R is ^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 is ^3’ Independently C _1-4 An alkyl group;

f is an integer from 1 to 4;

h is 0 or 1; and is also provided with

Y is COO, SO ₃ 、OPO(OR ^5' ) O OR P (O) (OR) ^5' ) O, where R is ^5’ Is H or C _1-4 Alkyl residues.

The zwitterionic surfactant may be a compound 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 ^- ；or

R ^1′ -CO-NH-(CH ₂ ) ₃ -N ⁺ (CH ₃ ) ₂ -CH ₂ CH(OH)CH ₂ SO ₃ ^- 。

in some embodiments, the surfactant is selected from the group consisting of almond oil amide propyl betaine (almondamidopropyl betaine), wild apricot oil amide propyl betaine (apricotamidopropyl betaine), avocado oil amide propyl betaine (avocadamidopropyl betaine), babassu oil amide propyl betaine (babassuamidopropyl betaine), behenamide propyl betaine (behenamidopropyl betaine), behenyl betaine (behenyl betaine), canola oil amide propyl betaine (canolamidopropyl betaine), octanoyl/decyl amide propyl betaine (capryl/capramidopropyl betaine), carnitine (carnitine), cetyl betaine (cetyl betaine), cocoamidoethyl betaine (cocamidoethyl betaine), cocoamidopropyl betaine (cocamidopropyl betaine), cocoamidopropyl hydroxysulfobetaine (cocomidopropyl betaine), coco betaine (coco betaine), coco hydroxysulfobetaine (coco hydroxysultaine), coco/oleamide propyl betaine (coco/oleamidopropyl betaine), coco sultaine (coco betaine), decyl/decyl betaine (capryl/capramidopropyl betaine), diglycol betaine (34), diglycol ethyl glycinate (37-glycine (37-38), diglycol glycinate (34), diglycol glycinate (37-ethyl glycinate), and triglycine (38-dihydroxyethyl glycinate (38) Isostearamidopropyl betaine (isostearamidopropyl betaine), lauramidopropyl betaine (lauramidopropyl betaine), laurylbetaine (laurylbetaine), laurylsulfobetaine (lauryl hydroxysultaine), laurylsulfobetaine (laurylsulfaine), cow's amidopropyl betaine (milk amidopropyl betaine), cow's amidopropyl betaine (milkamidopropyl betaine), nutmegamidopropyl betaine (myristamidopropyl betaine), nutmeg amidopropyl betaine (sesamidopropyl betaine), soybean amidopropyl betaine (oleamidopropyl betaine), oleamidopropylsultaine (oleamidopropylhydrofiltaine), oleyl betaine (oleyl betaine), olive oil amidopropyl betaine (olivamidopropyl betaine), palm oil amidopropyl betaine (palmamidopropyl betaine), palm amidopropyl betaine (palmitamidopropyl betaine), palm acyl carnitine (palmitoyl carnitine), palm kernel amidopropyl betaine (palm kernel amidopropyl betaine), polytetrafluoroethylene acetoxypropyl betaine (polytetrafluoroethylene acetoxypropyl betaine), ricinoleic amidopropyl betaine (ricinoleamidopropyl betaine), sesame amidopropyl betaine (sesamidopropyl betaine), soybean amidopropyl betaine (soyamidopropyl betaine), stearamidopropyl betaine (epoxypropyl betaine) (4872), stearamidopropyl betaine (epoxypropyl betaine) (oleamide, oleamide propyl betaine (oleamide propyl betaine) (4872), stearamidopropyl betaine (oleamide propyl betaine) (4872), undecanoamidopropyl betaine (undecylenamidopropyl betaine) and wheat germ amidopropyl betaine (wheat germ amidopropyl betaine). In some embodiments, the surfactant is lauryl betaine.

In some embodiments, the surfactant may be an anionic surfactant compound of the formula:

R ^1′ [CO-X(CH ₂ ) _j ] _g -(CH ₂ ) _f -[CH(OH)CH ₂ ] _h -Y ^- wherein

R ^1’ Is saturated or unsaturated C _5-24 An alkyl group;

x is NH, NR ^4’ Wherein R is ^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 is ^3’ Independently C _1-4 An alkyl group;

f is an integer from 0 to 4;

h is 0 or 1; and is also provided with

Y is COO, SO ₃ 、OPO(OR ^5’ ) O OR P (O) (OR) ^5’ ) O, where R is ^5’ Is H or C _1-4 Alkyl, and wherein the anionic surfactant may be in an acidic form or in the sodium or potassium salt form thereof. In some embodiments, f=0. In some embodiments, f=1. In some embodiments, f=3. In some embodiments, f=4. In some embodiments, f=1. In some embodiments, Y is COO or SO ₃ . In some embodiments, R ^2’ And R is ^3’ Is methyl.

The anionic surfactant is a compound according to the formula:

R ^1’ -CO-N(CH ₃ )-CH ₂ -COO ^- the method comprises the steps of carrying out a first treatment on the surface of the Or (b)

R ^1’ -CO-N(CH ₃ )-CH ₂ -SO ₃ -, wherein

R ^1’ Is saturated or unsaturated C _5-24 An alkyl group. In some embodiments, R ^1’ May be saturated or unsaturated C _7-19 Alkyl or C _11-17 An alkyl group.

In some embodiments, the anionic surfactant may be selected from the group consisting of N-lauroyl sarcosine, sodium palmitoyl sarcosine, sodium stearoyl sarcosine, sodium N-methyl-N- (1-oxotetradecyl) -glycine, sodium caproyl sarcosine, sodium capryloyl sarcosine, N-methyl-N- (1-oxo-9-octadecen-1-yl) -glycine, sodium salt, sodium oleoyl sarcosine, and sodium linoleoyl sarcosine. In some embodiments, the anionic surfactant is N-lauroyl sarcosine.

In some embodiments, the polymeric dye conjugate comprises a binding partner conjugated to a polymeric dye having the structure of formula III:

wherein,,

each a is independently selected from aromatic comonomers and heteroaromatic comonomers;

each optional M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers (bandgap-modifying monomer), optionally substituted ethylenes, and ethynylenes;

each optional L is a linker moiety;

G ¹ and G ² Each independently selected from unmodified polymer ends and modified polymer ends;

a. c and d define mole% of each unit, which may be uniform or randomly repeating, and wherein each a is 10% to 100% mole%, each c is 0 to 90% mole%, and each d is 0 to 25% mole%;

each b is independently 0 or 1;

and each m is an integer from 1 to about 10,000.

In some embodiments, a comprises a DHP moiety. In some embodiments, a comprises a fluorene moiety. In some embodiments, a comprises a DHP moiety and a fluorene moiety.

In some embodiments, the polymer dye conjugate is a polymer of formula I:

Wherein the method comprises the steps of

Each X is independently C or Si;

each Y is independently CR ¹ R ² Or SiR ¹ R ² ；

Each R ¹ Independently is an alkylammonium salt, an alkoxyammonium salt, an oligoether ammonium salt, an alkylsulfonate, an alkoxysulfonate, an oligoether sulfonate, an oligoether sulfonamide, or a water soluble moiety:

each R ² Independently is H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero) aryloxy, aryl, (hetero) arylamino, PEG groups, alkylammonium salts, alkoxyammonium salts, oligoether ammonium salts, alkylsulfonates, alkoxysulfonates, oligoether sulfonates, oligoether sulfonamides, or water-soluble moieties:

each R ³ Independently selected from H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero) aryloxy, aryl, (hetero) arylamino, and PEG groups;

each Z is independently selected from C, O and N;

each Q is independently selected from a bond, NH, NR ⁴ And CH (CH) ₂ The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

Each subscript n is independently an integer of from 0 to 20;

each M is a unit capable of varying the band gap of the polymer and is uniformly or randomly distributed along the polymer backbone; l is a linker; g ¹ And G ² Each independently selected from hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen substituted aryl, silyl, diazonium salt, triflate (triflate), acetoxy, azide, sulfonate (sulfonate), phosphate (phospho), boric acid substituted aryl, borate, boric acid, optionally substituted Dihydrophenanthrene (DHP), optionally substituted fluorene, aryl or heteroaryl, which is terminated with one or more side chains conjugated with a functional group selected from the group consisting of: amines, carbamates, carboxylic acids, carboxylic esters, maleimides, activated esters, N-hydroxysuccinimide groups, hydrazines, azides, alkynes, aldehydes, thiols, and protected groups thereof. In some embodiments, L is an aryl or heteroaryl group uniformly or randomly distributed along the polymer backbone and substituted with one or more side chains terminated with a functional group selected from the group consisting of: amines, carbamates, carboxylic acids, carboxylic esters, maleimides, activated esters, N-hydroxysuccinimides, hydrazines, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof; a. c and d define the mole% of each unit within the structure, each of which may be uniform or randomly repeating, and wherein a is 10% to 100% mole%, c is 0 to 90% mole%, and each d is 0 to 25% mole%; each b is independently 0 or 1; m is an integer from 1 to about 10,000; and each n is independently an integer from 1 to 20.

The binding partner may be a molecule or a molecular complex capable of specifically binding to the target analyte. The binding partner may be a protein, an affinity ligand, an antibody or an antibody fragment. In some embodiments, the binding partner is selected from the group consisting 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.

Compositions are provided comprising a polymer dye conjugate; an aqueous buffer; zwitterionic or anionic surfactants. The composition may comprise a concentration of zwitterionic surfactant or anionic surfactant below a critical micelle concentration (critical micellar concentration, CMC). In some embodiments, the concentration of the surfactant may be 0.05% to 0.25% (w/v), 0.06% to 0.20% (w/v), or 0.08% to 0.16% (w/v). The aqueous buffer may comprise further additives selected from the group consisting of protein stabilizers, preservatives and further surfactants. The composition may exhibit reduced non-specific binding of the polymer dye conjugate to white blood cells in a sample after exposure to the blood sample and flow cytometry analysis. The non-specific binding reduction is comparable to the same composition in the absence of zwitterionic or anionic surfactants. The white blood cells may be selected from monocytes and granulocytes.

Drawings

Fig. 1A shows a graph 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 absorption spectra of fluorene (Fl-Fl) polymer and dihydrophenanthrene (DHP-DHP) polymer. The DHP-DHP polymer (black curve) exhibits a maximum lambda value (lambda max) at 390 and 410nm, while the Fl-Fl (gray curve) polymer exhibits a maximum lambda value (lambda max) at about 400 nm.

Fig. 2 shows the following flow cytometry punctiform charts: undyed blood cells (upper panel); blood cells stained with polymer dye and without surfactant (bottom left panel); and blood cells stained with a composition comprising a polymeric dye and a surfactant (bottom right). Use of fluorescence without antibodiesThe photopolymer dye SN v605 stained the blood sample and was analyzed in a flow cytometer. The polymeric dye showed non-specific binding to monocytes/granulocytes without surfactant (bottom left). In the presence of EMPIGENThe polymeric dye showed significantly reduced non-specific binding to monocytes/granulocytes (bottom right).

Fig. 3 shows a dot pattern for: blood cells without polymer dye conjugate (upper panel) and blood cells stained with SN 605-CD20 conjugate with surfactant (lower left panel) and without surfactant (lower right panel). In the case of using a surfactant, the percentage of nonspecifically bound granulocytes decreases (check point "P2" gate in the dot pattern). Furthermore, the functional aspects of the conjugates were not altered (checking for "P1" gating in the dot plots) as the percentage of positive populations was similar in both cases.

Fig. 4 shows a bar graph of median fluorescence intensity (Median Fluorescence Intensity, MDFI) values for monocytes for two batches of polymer dye conjugate (SN v605-CD 20) in the presence and absence of surfactant, as compared to unstained monocytes (autofluorescence). In the presence of surfactant, non-specific interactions on monocytes were significantly reduced for both the Lot-1 and Lot-2SN605 cd20 conjugates.

Fig. 5 shows a bar graph of MdFI values of granulocytes in the presence and absence of surfactant for two batches of polymer dye conjugate (SN v605-CD 20) compared to unstained granulocytes (autofluorescence). In the presence of surfactant, nonspecific interactions on granulocytes were reduced for both the Lot-1 and Lot-2sn605 cd20 conjugates.

Fig. 6 shows the following dot pattern: blood cells without polymer dye conjugate (upper left) and in the presence of EmpigenBlood cells stained with SN v786-CD103 conjugate with surfactant (bottom left) and without surfactant (top right). The dot plot compares one of the claimed polymers with BV786-CD103 tandem fluorescent dye (bottom right) (available from Becton Dickinson). The percentage of non-specifically bound granulocytes and monocytes decreased in the presence of surfactant (checking for gating "P1" and "P2" in the dot plots, respectively).

Fig. 7 shows a bar graph of MdFI values of monocytes in the presence and absence of surfactant for two batches of polymer dye conjugate (SN v786-CD 103) compared to unstained monocytes (autofluorescence). In the presence of surfactant, the non-specific binding of the polymer conjugate to monocytes was significantly reduced for both batches of polymer dye conjugate.

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

FIG. 9 shows a dot-plot of blood cells stained with SN v605-CD20 conjugate with and without surfactant (upper panel), wherein the lower left panel is the nonionic surfactant Tween-20 and the lower right panel is the nonionic surfactant Pluronic F-68. In the case of using the non-ionic surfactants Tween-20 and Pluronic F-68, the percentage of non-specifically bound monocytes was not reduced (check the "non-specific monocytes" in the dot pattern).

Fig. 10 shows the following dot plots with blood cells without dye conjugate (upper left plot); blood cells stained with SN v605-CD20 conjugate in the presence of BSA (upper right panel), oxidized BSA (lower left panel) and BSA-Cy5-oX (lower right panel). In the case of protein blockers, the percentage of non-specifically bound monocytes and granulocytes was not significantly reduced (checking for "P1" gating in the dot-pattern).

Fig. 11 shows three graphs, each showing the effect of surfactant concentration on negative mononuclear cells (MFI) in unstained and stained samples of Donor1 (Donor 1, D1) and Donor 2 (Donor 2, D2) blood samples stained with specific SN v428CD19 (fig. 11, upper graph), SN v428CD 22 (fig. 11, lower graph) and SN v428CD 25 (fig. 11, middle graph). SN conjugate in the presence of 0.06% to 0.20% EmpigenShows a lower non-specific monocyte interaction than in the absence of surfactant.

FIG. 11 (follow) shows three graphs, each showing the effect of surfactant concentration on negative mononuclear cells (MFI) in unstained and stained samples of donor1 (D1) and donor 2 (D2) blood samples stained with specific SN v428CD19 (FIG. 11, upper graph), SN v428CD 22 (FIG. 11, lower graph) and SN v428CD 25 (FIG. 11, middle graph), data shown as Empigen-free Negative mononuclear cell MFI (in%) of the samples. Samples stained with BD Polymer dye conjugates were in the presence of 0.06% to 0.20% Empigen->Shows a lower non-specific monocyte interaction than in the absence of surfactant.

FIG. 12 (nine graphs) shows surfactant concentration versus absence of EmpigenNegative granulocytes (upper three panels), positive lymphocytes (middle panel) and positive lymphocytes (in%) of the samples (lower three panels). Shows unstained and stained blood samples from donor 1 (D1) and donor 2 (D2). The polymer dye conjugates for each of CD19 BD, CD25 BD and CD22 BD showed slightly lower negative interactions on granulocytes in the presence of 0.06% to 0.20% surfactant than in the absence of surfactant (top three figures). For each of the CD19 BD, CD25 BD and CD22 BD polymer dye conjugates, the positive lymphocyte data were similar or slightly higher in the presence of surfactant compared to the absence of surfactant (middle three panels and bottom three panels).

FIG. 13 shows that there are 0.06%, 0.12% and 0.2% Empigen in the absence of surfactant Dot plots of SS/FL9 staining patterns for SN v428 CD19 lot#d19-094 polymer dye conjugate at 0.5 μg/time with surfactant, and CD19 BV-421 conjugate (Becton Dickinson) at its commercial dose for donor 1 (upper panel) and donor 2 (lower panel) blood samples.

FIG. 14 shows a plot of the SS/FL9 staining pattern of 0.06%, 0.12% and 0.2% surfactant without surfactant for the SN v428 CD25 Lot#D19-107 polymer dye conjugate at 0.5 μg/time and CD25 BV-421 conjugate (Becton Dickinson) at its commercial dose for donor 1 (upper panel) and donor 2 (lower panel) blood samples.

FIG. 15 shows plots of the polymer dye conjugates at 0.5 μg/time tested against SN v428 CD22 Lot#D19-109 without surfactant, 0.06%, 0.12% and 0.2% surfactant, and CD22 BV-421 conjugate (Becton Dickinson) at its commercial dose versus donor 1 (upper panel) and donor 2 (lower panel) blood samples.

Fig. 16 shows a plot of the percentage of dead cells up to 0.2% surfactant. In the absence of EMPIGEN(negative control) and 0.06%, 0.12% and 0.2% EMPIGEN- >CD19-SN v428D19-094 was tested on donor 1 and donor 4 whole blood samples stained with 7-ADD to evaluate the percentage of dead cells in each case. Whole blood samples that had been stored for more than 24 hours were added as positive controls for 7-AAD staining (left panel, 12% dead cells). When compared to the sample without surfactant, up to 0.2% EMPIGEN->Without significantly increasing the percentage of dead cells.

Fig. 17A shows a dot plot of a peripheral blood sample without a monochromatic conjugate, demonstrating the absence of populations in the cd20+ gate.

FIG. 17B shows the composition of BSA, sodium azide, pluronic ^TM F-68 (PF-68) and EmpigenPositive control dot pattern of peripheral blood samples in the presence of CD20-SN v605 single color conjugate in buffer composition as additive. When compared to the negative control stippling (FIG. 17C), the% populations in gating "Mons non-specific binding" and "Grans non-specific binding" were each significantly reduced, indicating Empigen->Effectiveness in eliminating or reducing non-specific binding to monocytes and granulocytes.

FIG. 17C shows a negative control dot pattern of peripheral blood samples in the presence of CD20-SN v605 single-color conjugate in a buffer composition containing only BSA, PF-68 and sodium azide as additives.

FIG. 17D shows a plot of test spots of peripheral blood samples in the presence of CD20-SN v605 single color conjugate in a buffer composition containing BSA, sodium azide, PF-68 and NLS (0.16% w/v) as additives.

FIG. 17E shows a plot of test spots of peripheral blood samples in the presence of CD20-SN v605 single color conjugate in a buffer composition containing BSA, sodium azide, PF-68 and NLS (0.08% w/v) as additives.

Detailed Description

Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in the accompanying drawings and examples. Although the disclosed subject matter will be described in connection with the enumerated claims, it should be understood that the exemplary subject matter is not intended to limit the claims to the disclosed subject matter.

General disclosure

The present disclosure relates generally to compositions and methods of using compositions to detect an analyte in a sample, the compositions comprising at least one surfactant and at least one polymer dye conjugated to a binding partner (e.g., an antibody), such as a fluorescent polymer dye conjugated to a binding partner. More specifically, the present disclosure relates to methods for reducing or eliminating non-specific binding of at least one polymer dye conjugate in a biological sample (e.g., a blood sample), the methods comprising: contacting the at least one polymer dye conjugate with at least one zwitterionic or anionic surfactant prior to, during, or after the contacting of the polymer dye conjugate with a biological sample (e.g., a blood sample), the contacting resulting in reduced non-specific binding of the at least one polymer dye conjugate to cells (e.g., leukocytes) in the blood sample. The surfactant may be added to the blood sample prior to the contacting. The surfactant may be added to the polymer dye conjugate prior to contact with the biological sample.

Definition of the definition

Abbreviations used herein have their conventional meaning in the chemical and biological arts.

Unless the context clearly indicates otherwise, nouns without quantitative word modifications represent one or more.

The term "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items. The term "room temperature" refers to 18 to 27℃ unless otherwise specified

The term "percent" or "%" refers to weight percent unless otherwise indicated.

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 component in a sample. Analytes may 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 that is capable of specifically binding to an analyte. The binding partner may be any of a number of different types of molecules, including antibodies or antigen binding fragments thereof, or other proteins, peptides, polysaccharides, lipids, nucleic acids or nucleic acid analogues, such as oligonucleotides, aptamers or PNA (peptide nucleic acid) (peptide nucleic acids).

The term "CD" refers to cluster of differentiation (Cluster of differentiation).

The term "compensation" in flow cytometry is a mathematical method for correction of fluorescence extravasation (spectral overlap of multiparameter flow cytometry data). For example, compensation may be performed by removing the signal of any given fluorescent dye from all detectors except the detector dedicated to measuring that dye. Since fluorescent dyes can have a wide range of spectra, they can overlap, causing undesirable confusion during data analysis.

The term "labeled binding partner" refers to a binding partner conjugated to a dye. The term "reactant solution" refers to a solution comprising labeled binding partners. In some embodiments, the reactant solution may include stabilizers, salts, buffers, surfactants, and/or other reagents in addition to the labeled binding partners.

The term "linker" or "linker" refers to a linking moiety that connects two groups and has a backbone of 100 atoms or less in length. The linker or linker may be a covalent bond linking two groups or a chain of 1 to 100 atoms in length, for example a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, wherein the linker may be linear, branched, cyclic or a single atom. In some embodiments, the linker is a branched linker, which refers to a linking moiety that links three or more groups. In some cases, one, two, three, four, or five or more carbon atoms of the linker backbone may be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. In some embodiments, the linker backbone comprises a linking functional group, such as an ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. The bonds between the 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 may comprise one or more substituents, for example having an alkyl, aryl or alkenyl group. The linker may include, but is not limited to, polyethylene glycol, ether, thioether, tertiary amine, alkyl, which may be straight or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1-dimethylethyl (t-butyl), and the like. The linker backbone may comprise a cyclic group, such as aryl, heterocyclic or cycloalkyl, wherein 2 or more atoms of the cyclic group, such as 2, 3 or 4 atoms, are comprised in the backbone. The linker may 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 the linker moiety may be attached to "L", as taught in U.S. published application No.2019/0144601, which is incorporated herein by reference in its entirety. The linker moiety may comprise a sulfonamide, disulfonamide, selenoamide, sulfenamide, disulfonamide, amide, seleniinamide, phosphonamide, phosphinamide, phosphonamic acid ester, or secondary amine.

As described therein, and as each pertains to a linker moiety, the term "sulfonamide" refers to moiety-S (O) ₂ NR-; the term "disulfonamides"Refers to part-S (O) ₂ NRS(O) ₂ -; the term "selenoamide" refers to part-Se (O) ₂ NR-; the term "sulfinamide" refers to the moiety-S (O) NR-; the term "disulfinamide" refers to the moiety-S (O) NRS (O) -; the term "selenimide" refers to the moiety-Se (O) NR-; the term "phosphonamide" refers to a moiety-NR-PR (O) NR-; the term "phosphinamide" refers to the moiety-PR (O) NR-; and the term "phosphonimidoester" refers to a moiety-O-PR (O) NR-; and the term "sultam" refers to cyclic sulfonamides (e.g., wherein the R group is bonded to the sulfur atom through an alkylene moiety); wherein for each term, the R groups are independently H, alkyl, haloalkyl or aryl.

The term "terminal" as used herein refers to the end on the conjugated polymer chain, which may comprise a functional group that provides bioconjugation. In some cases, such functionality is referred to as terminal linkers. The terminal may be, for example, hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen substituted aryl, silyl, diazonium salt, trifluoromethanesulfonic acid, acetoxy, azide, sulfonate, phosphate, borate substituted aryl, borate, boric acid, optionally substituted Tetrahydropyrene (THP), optionally substituted fluorene, optionally substituted Dihydrophenanthrene (DHP), aryl or heteroaryl, substituted with one or more side chains terminated with functional groups selected from the group consisting of: amines, carbamates, carboxylic acids, carboxylic acid esters, maleimides, activated esters, N-hydroxysuccinimide groups, hydrazines, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof for conjugation to substrates or binders.

The term "MdFI" or "MdFI" refers to the median fluorescence intensity (Median fluorescence intensity).

The term "% recruitment" refers to the number of gating cells of the relevant population.

The term "multiplex" herein refers to assays or other analytical methods that can simultaneously analyze multiple analytes.

The term "PEG" refers to polyethylene glycol or poly (ethylene glycol). The numbers following "PEG" refer to average molecular weights, where Mw refers to weight average molecular weights, and Mn refers to number average molecular weights.

The term "PBS" refers to phosphate buffered saline, which is an aqueous buffer, and may comprise sodium chloride, disodium hydrogen phosphate, potassium chloride, and potassium dihydrogen phosphate. For example, PBS may contain milliQ water or deionized water, 137mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ 、1.8mM KH ₂ PO ₄ . 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 (side scatter).

The acronym "WBC" refers to white blood cells.

A "dye" is a moiety that provides a detectable signal, which can be directly or indirectly linked to or incorporated into a binding partner. Dyes used in the present disclosure may be colored, fluorescent, or luminescent, and are typically detected by a detector (e.g., PMT or APD) in a flow cytometer. The fluorescent dye may be a monomer or a polymer. The fluorescent dye may be a fluorescent polymer dye. Polymeric dyes are particularly useful for analysis of chemical and biological targets. Since they contain multiple chromophores, they are highly responsive optical reporters and efficient light absorbers. Fluorescent polymer dyes suitable for use in the present disclosure are described herein, for example in US 2019/0144601 and US 2020/0190253. Some examples of polymeric dyes include, but are not limited to, conjugated polymers having chromophore repeat units, aggregates of conjugated molecules, luminescent dyes linked to saturated polymers through side chains, semiconductor quantum dots, and dendritic structures. The polymer and monomer dyes disclosed in U.S. Pat. nos. 7,214,489, 8,354,239, 8,575,303 are also useful in the present invention.

The term "ammonium" as used herein refers to a compound having the formula NHR ₃ ⁺ Wherein each R group is independently hydrogen or a substituted or unsubstituted alkyl, aryl, arylalkyl or alkoxy group. Preferably, each R group isHydrogen.

As used herein, "oligoether" is understood to mean an oligomer comprising structural repeat units having ether functionality. "oligomer" as used herein is understood to be a molecule comprising one or more identifiable structural repeat units of the same or different formula.

The term "sulfonate functional group" or "sulfonate" as used herein refers to the free sulfonate anion (-S (=o) ₂ O-) and salts thereof. Thus, the term sulfonate encompasses sulfonates such as sodium sulfonate, lithium sulfonate, potassium sulfonate, and ammonium sulfonate.

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

The term "alkyl" as used herein refers to a straight or branched chain saturated aliphatic group having the indicated number of carbon atoms. For example, C ₁ -C ₆ Alkyl groups include, but are 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. The alkyl group may comprise any number of carbons, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, and 5 to 6. Alkyl groups are typically monovalent, but may also be divalent, for example when the alkyl groups join two moieties together.

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

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

The term "halogen" as used herein 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. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like. Alkoxy groups may be further substituted with a variety of substituents described herein. For example, an alkoxy group may be substituted with a halogen to form a "halo-alkoxy" group.

The term "olefin" as used herein refers to a straight or branched hydrocarbon having at least one double bond. Some examples of alkenyl groups include, but are not limited to, ethenyl, 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. Alkenyl groups are typically monovalent, but may also be divalent, for example when alkenyl groups join two moieties together.

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

The term "aryl" as used herein refers to a monocyclic or fused bicyclic, tricyclic or larger collection of aromatic rings containing 6 to 16 ring carbon atoms. For example, aryl may be phenyl, benzyl or naphthyl, preferably phenyl. "arylene" means a divalent group derived from an aryl group. The aryl group may be selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino-alkyl, trifluoromethyl, alkylenedioxy and oxo-C ₂ -C ₃ -one, two or three groups in the alkylene group are mono-, di-or tri-substituted; all of these substituted aryl groups are optionally further substituted, for example as defined above; or aryl may be 1-or 2-naphthyl; or 1-or 2-phenanthryl. Alkylene dioxy is a divalent substituent attached to two adjacent carbon atoms of a phenyl group, such as methylene dioxy or ethylene dioxy. oxygen-C ₂ -C ₃ Alkylene is also a divalent substituent attached to two adjacent carbon atoms of the phenyl group, such as oxyethylene or oxypropylene. oxygen-C ₂ -C ₃ An example of an alkylene-phenyl group is 2, 3-dihydrobenzofuran-5-yl.

Preferred aryl groups are naphthyl, phenyl or phenyl, in particular phenyl, which is mono-or disubstituted by alkoxy, phenyl, halogen, alkyl or trifluoromethyl, and in particular phenyl, which is mono-or disubstituted by alkoxy, halogen or trifluoromethyl.

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

The term "polyethylene glycol" or "PEG" as used herein refers to a polymer based on the formula- (CH) ₂ -CH ₂ -O-) _n -or derivatives thereof describe a family of biocompatible water-soluble linear polymers of ethylene glycol monomer units. In one placeIn 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, 15 or less, for example, 3 to 15 or 10 to 15. It should be understood that the PEG polymeric groups may be of any convenient length and may contain a variety of end groups and/or other substituents including, but not limited to, alkyl, aryl, hydroxyl, amino, acyl, carboxylic acid, carboxylate, acyloxy, and amido end groups and/or substituents.

The term "heteroaryl" as used herein refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, wherein 1 to 4 ring atoms are heteroatoms, each N, O or S. For example, heteroaryl groups include pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, Azolyl, iso->An oxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl or any other substituted group, in particular a group mono-or di-substituted for example by alkyl, nitro or halogen. Pyridyl represents 2-, 3-or 4-pyridyl, advantageously 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, benzothiopyranyl (benzothiopyranyl) preferably represents 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl preferably represents 2-or 4-thiazolyl, and most preferably 4-thiazolyl. The triazolyl group is preferably 1-, 2-, or 5- (1, 2, 4-triazolyl). The tetrazolyl group is preferably a 5-tetrazolyl group.

Preferably, heteroaryl is pyridinyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, iso-arylAzolyl, triazolylTetrazolyl, pyrazolyl, imidazolyl, thienyl, furyl, benzothiazolyl, benzofuryl, isoquinolyl, benzothienyl, and,An oxazolyl, indazolyl or any substituted group, in particular a mono-or di-substituted group.

Similarly, the substituents for aryl and heteroaryl are different and are selected from: -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(NH ₂ )═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 groups ranging in number from zero to the total number of open valencies on the aromatic ring system; and wherein R ', R ' and R ' are independently selected from hydrogen, (C) ₁ -C ₅ ) Alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl) - (C) ₁ -C ₄ ) Alkyl and (unsubstituted aryl) oxy- (C ₁ -C ₄ ) An alkyl group.

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

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

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

The term "carbamate" as used herein refers to a compound having the structure-NR "CO ₂ A functional group of R ', wherein R ' and R ' are independently selected from hydrogen, (C) ₁ -C ₈ ) Alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl) - (C) ₁ -C ₄ ) Alkyl and (unsubstituted aryl) oxy- (C ₁ -C ₄ ) An alkyl group. Some examples of carbamates include t-Boc, fmoc, benzyloxy-carbonyl, alloc, methyl carbamate, ethyl carbamate, 9- (2-sulfo) fluorenylmethylcarbamate, 9- (2, 7-dibromo) fluorenylmethylcarbamate, tbfmoc, climoc, bimoc, DBD-Tmoc, bsmoc, troc, teoc, 2-phenethylcarbamate, adpoc, 2-chloroethylcarbamate, 1-dimethyl-2-haloethylcarbamate, DB-t-BOC, TCBOC, bpoc, t-Bumeoc, pyoc, bnpeoc, V- (2-pivaloylamino) -1, 1-dimethylethylcarbamate, npSSPeoc.

The term "carboxylate" as used herein refers to a conjugate base of a carboxylic acid, which may 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 in peptide chemistry for promoting easy condensation of a carboxyl group with a free amino group of an amino acid derivative. Descriptions of these carboxyl-activating groups can be found in the general textbooks of peptide chemistry; for example, k.d. kopple, "Peptides and Amino Acids", w.a. benjamin, inc., new York,1966, pages 50 to 51 and e.schroder and k.lubke, "The Peptides"; vol.1, academic Press, new York,1965, pages 77 to 128.

The terms "hydrazine" and "hydrazide" refer to compounds that contain singly-bound nitrogen, one of which is a primary amine functional group.

The term "aldehyde" as used herein refers to a compound having a —cho group.

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

The term "silyl" as used herein refers to Si (R ^z ) ₃ Wherein each R is ^z Independently an alkylaryl or other carbon containing atomic group.

The term "diazonium salt" as used herein refers to a salt having the structure R-N ₂ ⁺ X ^- Wherein R can be any organic residue (e.g., alkyl or aryl) and X is an inorganic or organic anion (e.g., halogen).

The term "triflate" also known as triflate (triflate) is a compound having the formula CF ₃ SO ₃ Is a group of (2).

The term "boric acid" as used herein refers to structure-B (OH) ₂ . Those skilled in the art recognize that boric acid may be present as a borate at various stages of quencher synthesis. Boric acid is meant to include such esters. The term "borate" or "borate" as used herein means a compound containing-B (Z ¹ )(Z ² ) A partial compound wherein Z ¹ And Z ² Together form a moiety in which the atom attached to the boron in each case is an oxygen atom. The borate moiety may be a 5-membered ring. The borate moiety may be a 6 membered ring. The borate moiety may be a mixture of 5-membered and 6-membered rings.

Values expressed in a range format are to be construed in a flexible manner to include not only the values explicitly recited as the limits of the range, but also to include all the individual values or sub-ranges encompassed within that range as if each value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. Unless otherwise indicated, the recitations "about X to Y" and "about X to about Y" have the same meaning. Also, unless otherwise indicated, the statement "about X, Y or about Z" has the same meaning as "about X, about Y or about Z".

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

"SN" as used herein refers to the "Super Nova" dye commercially available from Beckman Coulter, inc.

"EMPIGEN" as used herein"refers to a zwitterionic surfactant (CAS No. 66455-29-6) comprising N, N-dimethyl-N-dodecyl betaine at about 30% betaine concentration in an aqueous solution. />

The term "about" as used herein may allow for some degree of variability in a value or range, for example, within 10%, within 5% or within 1% of a specified value or specified range limit.

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 on a cell or to a target analyte targeted by the antibody or binding partner.

"non-specific binding" as used herein refers to the binding of an antibody or other binding partner (e.g., in a polymer conjugated dye) to a cell or sample component that does not contain an epitope that is targeted by the antibody or other binding partner. 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" non-specific binding of a polymer dye conjugate may refer to a reduction in average fluorescence intensity (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%) when "negative" (e.g., negative granulocytes, monocytes, and lymphocyte populations) relative to% when no surfactant is used. In other words, the% reduction in at least one of the background staining of monocytes, granulocytes and lymphocytes is reduced by 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%) relative to% without the surfactant.

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

The term "substantially free" or "substantially free" as used herein means less than about 1%, 0.5%, 0.1%, 0.05%, 0.001%, or less than about 0.0005% or less, about 0%, below a limit of quantitation, below a detectable limit or 0%.

In this document, unless otherwise indicated, nouns having no quantitative modifications are indicative of one or more than one. The term "or/and" is used to refer to a non-exclusive "or/and" unless otherwise specified. Also, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description and not of limitation. The use of any section headings is intended to aid reading this document and should not be construed as limiting. In addition, information related to chapter titles may appear inside or outside the particular chapter. In addition, all publications, patents, and patent documents mentioned in this document are incorporated by reference in their entirety as if individually incorporated by reference. If the usage between this document and those documents so incorporated by reference is inconsistent, the usage in the incorporated references should be considered as a complement to the usage of this document; for contradictory inconsistencies, the usage in this document shall control.

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

In certain embodiments, dye compositions are provided that comprise at least one polymeric dye conjugated to at least one suitable anionic surfactant.

In some embodiments, methods for reducing or eliminating non-specific binding of at least one polymer dye conjugate to cells in a biological sample (e.g., a blood sample) are provided, the methods comprising contacting at least one polymer dye conjugate with at least one zwitterionic and/or anionic surfactant before, during, and/or after the at least one polymer dye conjugate is contacted with the biological sample. The steps may be performed in any order, other than the explicitly recited times or sequence of operations, without departing from the principles of the invention. Furthermore, unless an explicit claim language recites a particular step to be performed separately, the particular step may be performed concurrently. For example, the claimed step of performing X and the claimed step of performing Y may be performed simultaneously in a single operation, and the resulting process would fall within the literal scope of the claimed method. Thus, in some cases, the at least one polymer dye conjugate may be contacted with the at least one zwitterionic surfactant or anionic surfactant prior to the at least one polymer dye conjugate being contacted with the blood sample. In some cases, the at least one polymer dye conjugate may be contacted with the at least one zwitterionic surfactant or anionic surfactant at the same time as the at least one polymer dye conjugate is contacted with the blood sample.

Surface active agent

Various types of surfactants have been explored for reducing or preventing non-specific interactions of polymer dye conjugates with biological samples.

Suitable surfactants may be zwitterionic or some anionic surfactant. Some examples of suitable surfactants include surfactants of 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 ^- Wherein R is ^1’ Is saturated or unsaturated C _5-24 Alkyl radicals, e.g. 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 An alkyl group; x is NH, NR ^4' Wherein R is ^4' Is C _1-4 Alkyl, O or S; j is an integer from 1 to 10, for example 2 to 5 and 3; g is 0 or 1, R ^2’ And R is ^3’ Each independently is C _1-4 Alkyl groups such as ethyl or methyl; hydroxy optionally substituted with hydroxyethyl or methyl; k is 0 or 1; f is an integer from 0 to 4, for example 0, 1, 2, 3 or 4; h is 0 or 1; and Y is COO, SO ₃ 、OPO(OR ^5' ) O OR P (O) (OR) ^5' ) O, where R is ^5’ Is H or C _1-4 Alkyl, and when k=0, the surfactant may be in an acidic form or a sodium or potassium salt thereof.

In accordance with the present disclosure, the surfactant may be present in a concentration of about 0.05% to about 0.25%, about 0.06% to about 0.2%, or about 0.08% to about 0.16% (w/v) in a buffer or other suitable aqueous composition.

Suitable zwitterionic surfactants that may be used according to the methods described herein include betaine zwitterionic surfactants such as alkyl betaines, alkyl amidobetaines, amidoazo betaines, sulfobetaines (INCI sulfobetaines), and phosphobetaines.

Some 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 ^- the method comprises the steps of carrying out a first treatment on the surface of the And

R ^1' -CO-NH-(CH ₂ ) ₃ -N ⁺ (CH ₃ ) ₂ -CH ₂ CH(OH)CH ₂ SO ₃ ^- 。

some examples of suitable betaines and sulfobetaines are as follows (specified according to INCI): almond oil amide propyl betaine, wild apricot oil amide propyl betaine, avocado oil amide propyl betaine, babassu oil amide propyl betaine, behenamide propyl betaine, behenyl betaine, canola oil amide propyl betaine, octanoyl/decyl amide propyl betaine, carnitine, cetyl betaine, coco amide ethyl betaine, coco amide propyl hydroxysulfobetaine, coco oil betaine, coco hydroxysulfobetaine, coco/oil amide propyl betaine, coco sulfobetaine, decyl betaine, dihydroxyethyl oil glycine, dihydroxyethyl soybean glycine, dihydroxyethyl stearyl glycine, dihydroxyethyl tallow glycine, PG-betaine of dimethicone propyl, erucic acid amide propyl hydroxysulfobetaine, hydrogenated tallow betaine isostearamidopropyl betaine, lauramidopropyl betaine, lauryl hydroxysulfobetaine, laurylsulfobetaine, milk amidopropyl betaine myristamidopropyl betaine, myristyl betaine, oleamidopropyl hydroxysulfobetaine, oleamidobetaine, olive oleamidopropyl betaine palm oleyl amidopropyl betaine, palm acyl carnitine, palm kernel oleyl amidopropyl betaine, polytetrafluoroethylene acetoxypropyl betaine, ricinoleic amidopropyl betaine, sesame amidopropyl betaine, soybean amidopropyl betaine, stearamidopropyl betaine, stearamidobetaine, tallow amidopropyl betaine, tallow amidopropyl hydroxysulfobetaine, tallow betaine, tallow dihydroxyethyl betaine, undecylaminopropyl betaine and wheat germ amidopropyl betaine.

Suitable betaine zwitterionic surfactants may be N- (alkyl C) _10-16 ) -N, N-dimethylglycine betaine, N- (alkyl C) _12-14 ) -N, N-dimethyl betaine, N-dimethyl-N-dodecyl betaine, lauryl dimethyl betaine (also called lauryl betaine), myristyl sulfobetaine or N-hexadecyl-N, N-dimethyl-3-amino-1-propane sulfonate. Lauryl betaine can be used as EMPIGEN(Huntsman Corporation) is commercially available and has a CMC of 1.6 to 2.1mM (20 to 25 ℃). Myristylsulfobetaine (also known as N-tetradecyl-N, N-dimethyl-3-amino-1-propanesulfonate, DMMA) may be found under the trade name +>3-14 (Merck KGaA, darmstadt, germany) and CMC of 100 to 400. Mu.M. N-hexadecyl-N, N-dimethyl-3-amino-1-propane sulfonate (also known as 3-N, N-dimethylpalmitin) propane sulfonate, DMPA) can be given the trade name>3-16, and CMC of 10 to 60 μm. For example, coconut oil dimethyl betaine may be under the trade name AMONYL +.>Commercially available from Seppic; and lauryl betaine can be given the trade name EMPIGEN +.>Commercially available from Sigma-Aldrich. Another example of betaine is available under the trade name MIRATAINE +.>Lauryl-imino-dipropionate is commercially available from Rhodia.

In accordance with the present disclosure, the zwitterionic surfactant may be present in a buffer or other suitable aqueous composition in a range of about 0.06% to about 0.2% or about 0.08% to about 0.16%.

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

Some examples of suitable anionic surfactants include those of the general formula R ^1′ [CO-X(CH ₂ ) _j ] _g -(CH ₂ ) _f -[CH(OH)CH ₂ ] _h -Y ^- Wherein R is ^1’ Is saturated or unsaturated C _5-24 Alkyl radicals, e.g. C _8-18 Alkyl, saturated C _10-16 Alkyl or saturated C _12-14 An alkyl group; x is NH, NR ^4’ Wherein R is ^4’ Is C _1-4 Alkyl, O or S; j is an integer from 1 to 10, for example 2 to 5 and 3; g is 0 or 1; f is an integer from 0 to 4, for example 0, 1, 2, 3 or 4; h is 0 or 1; and Y is COO, SO ₃ 、OPO(OR ^5’ ) O OR P (O) (OR) ^5’ ) O, where R is ^5’ Is H or C _1-4 Alkyl, and wherein the anion is surface activeThe sex agent may be in the acidic form or its sodium or potassium salt form.

Suitable anionic surfactants may include the following structures:

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 x, wherein a = 1 to 8; b=0 to 2, and c=0 to 6, x=h, na, K.

Some examples of alkanoyl sarcosinates may include those of the formula and e.g. sodium or potassium salts thereof:

R ^1’ -CO-N(CH ₃ )-CH ₂ -COO-; and

R ^1’ -CO-N(CH ₃ )-CH ₂ -SO ₃ ^- wherein R is ^1’ May be saturated or unsaturated C _5-24 Alkyl, C _7-19 Alkyl or C _11-17 An alkyl group.

Some examples of suitable alkanoyl sarcosinates and acid or salt forms thereof include N-lauroyl sarcosine, sodium lauroyl sarcosinate, sodium palmitoyl sarcosinate, sodium stearoyl sarcosinate, sodium N-methyl-N- (1-oxotetradecyl) -glycine, 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 may be 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%.

The composition may be used in a flow cytometer and thus may contain additional components including, but not limited to, one or more of the following: any suitable carrier, stabilizer, buffer, salt, chelating agent (e.g., EDTA), or preservative. The composition may further comprise one or more additional surfactants in addition to the zwitterionic and/or anionic surfactants described herein. Some non-limiting examples of the one or more additional surfactants include polysorbate For example20 (polyoxyethylene sorbitol monolaurate) and +.>80 (polyoxyethylene sorbitol monooleate). The carrier may 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 include a formulation, such as a suspending, stabilizing and/or dispersing agent. The composition may also contain a buffer or pH adjuster, and typically the buffer is a salt prepared from an organic acid or base. Exemplary buffers include salts of organic acids such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; tris-tromethamine hydrochloride or phosphate buffer.

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

The stabilizing agent may be gelatin, a protein, typically derived from collagen obtained from a body part of an animal. Gelatin is brittle when dry and tacky when wet. It may also be referred to as hydrolyzed collagen, collagen hydrolysate, gelatin hydrolysate, hydrolyzed gelatin, and collagen peptide after gelatin has been hydrolyzed. Several types of gelatin are commercially available, including type A gelatin, type B gelatin,High purity type a gelatin and cold water fish gelatin.

The composition may also comprise any suitable preservative. The preservative may be an antioxidant, biocide or antimicrobial agent. The preservative may be an inorganic salt. The preservative may be sodium azide. The preservative 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%.

Polymeric dyes

In another embodiment, the composition may be used with a polymeric dye. The polymer dye may be a fluorescent polymer dye or a fluorescent polymer tandem dye. Polymeric dyes are particularly useful for analysis of chemical and biological target analytes. Since the polymeric dye contains multiple chromophores, it is a highly responsive optical reporter and an effective light absorber. The polymer dye conjugate may comprise any of the previously disclosed fluorescent polymer dyes or fluorescent polymer tandem dyes.

For example, the polymeric dye or tandem polymeric dye may be any dye disclosed in the following: PCT application No. wo 2017/180998; U.S. application Ser. No.2021/0047476; U.S. application Ser. No.2020/0190253; U.S. application Ser. No.2020/0147615; U.S. application Ser. No.2021/0108083; U.S. application Ser. No.2018/0224460; U.S. Pat. No.11,034,840; U.S. Pat. No.10,228,375; U.S. Pat. No.10,545,137B2; U.S. Pat. No.10,533,092; U.S. Pat. No.7,214,489; U.S. Pat. No.8,354,239; U.S. patent No.8,575,303, each of which is incorporated by reference as if fully set forth herein in its entirety. The polymer dye conjugate may have the structure of any of the water-soluble fluorescent polymer dyes disclosed in published U.S. application No.2020/0190253A1, which is incorporated by reference as if fully set forth herein in its entirety. The polymer dye conjugate may have the structure of any of the water-soluble fluorescent polymer dyes disclosed in published U.S. application No.2019/0144601, which is incorporated by reference as if fully set forth herein in its entirety.

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

In some embodiments, the polymeric dye or polymeric dye conjugate may comprise SuperNova ^TM Dyes (Beckman Coulter, inc.). SuperNova ^TM Polymers are a new generation of polymeric dyes that can be used in flow cytometer applications. The polymer dye or polymer dye conjugate may comprise SuperNova ^TM v428、SuperNova ^TM v605 or SuperNova ^TM v786(Beckman Coulter,Inc.)。SuperNova ^TM v428 has unique photophysical properties that when bound to an antibody or other binding partner, result in an extremely bright conjugate. For example, superNova ^TM v428 (SN v 428) (Beckman Coulter, inc.) is a polymeric dye that can be optimally excited by a violet laser (e.g. 405 nm) with an excitation maximum of 414nm and an emission peak of 428nm, and can be detected using a 450/50 bandpass filter or equivalent.

SuperNova ^TM v428 is one of the brightest dyes that can be excited by a violet laser and is therefore particularly suitable for assessing markers that express darkness. SuperNova ^TM The conjugated antibody may comprise an anti-CD 19 antibody-SuperNova ^TM v428, anti-CD 22 antibody-SuperNova ^TM v428, anti-CD 25 antibody-SuperNova ^TM v428 and anti-CD 38 antibody-SuperNova ^TM v428 antibody-polymer dye conjugate.

SuperNova ^TM v605 and SuperNova ^TM v786 (Beckman Coulter, inc.) is a tandem polymer dye derived from the core SuperNova ^TM v428 polymeric dye. Both have the same absorbance characteristics and the excitation maximum is at 414 nm. Due to SuperNova ^TM v605 and SuperNova ^TM The emission peaks of v786 were at 605nm and 786nm, respectively, so they could be best detected using 610/20 and 780/60nm bandpass filters of the flow cytometer. SuperNova ^TM v605 and SuperNova ^TM v786 may be conjugated with, for example, anti-CD 19 antibodies, anti-CD 22 antibodies, anti-CD 25 antibodies, and anti-CD 38 antibodies.

The polymer dye or polymer dye conjugate may comprise a polymer dye that is excitable by an ultraviolet ("UV") laser. The polymer dye or polymer dye conjugate may comprise a polymer dye that is excitable by a UV laser having a wavelength of 320nm to 380nm, 340nm to 360nm, 345nm to 356nm, or less than or equal to 380nm but greater than or equal to 320 nm. The polymer dye or polymer dye conjugate may comprise a UV excitable polymer dye. The UV excitable polymer dye or polymer dye conjugate may generally emit light at wavelengths of 380nm to 430nm, 406nm to 415nm, or less than or equal to 430nm but greater than or equal to 380 nm.

The polymeric dye or polymeric dye conjugate may comprise Brilliant Violet ^TM Dye [ ]Siriben Group Ltd.), e.g. Brilliant Violet 421 ^TM (excitation maximum 405nm, emission maximum 426 nm,450/50 filter), brilliant Violet 510 ^TM (excitation maximum 405nm, emission maximum 510nm,510/50 filter), brilliant Violet 570 ^TM (excitation maximum 405nm, emission maximum 570nm,585/42 filter), brilliant Violet 605 ^TM (excitation maximum 405nm, emission maximum 603nm,610/20 filter), brilliant Violet 650 ^TM (excitation maximum 405nm, emission maximum 640 nm,660/20 filter), briliant Violet 711 ^TM (excitation maximum 405nm, emission maximum 711nm,710/50 filter), brilliant Violet 750 ^TM (excitation maximum 405nm, emission maximum 750nm,780/60 filter), brilliant Violet 785 ^TM (excitation maximum 405nm, emission maximum 785nm,780/60 filter). The polymer dye or polymer dye conjugate may comprise Spark Violet ^TM 538 (BioLegend, inc.) (excitation maximum 405nm, emission maximum 538 nm).

The polymer dye or polymer dye conjugate may comprise a Super Bright dye (Invitrogen, thermoFisher Scientific). The Super Bright dye can be excited by a violet laser (405 nm). The Super Bright dye may be Super Bright 436 (excitation maximum 414nm, emission maximum 436nm,450/50 bandpass filter), super Bright 600 (emission maximum 600nm,610/20 bandpass filter), super Bright 645 (emission maximum 645nm,660/20 bandpass filter), or Super Bright 702 (emission maximum 702nm,710/50 bandpass filter).

The polymer dye or polymer dye conjugate may comprise BD Horizon Brilliant ^TM Purple polymer dye (Becton, dickinson and co., BD Life Sciences). The polymeric dye may be BD Horizon Brilliant ^TM BV421 (450/40 or 431/28 filters), BV480 (525/40 filters), BV510 (525/40 filters), BV605 (610/20 filters), BV650 (660/20 filters), BV711 (710/50 filters), BV786 (786/60 filters).

Polymeric dyes can be prepared synthetically by monomer polymerization, which results in the formation of highly conjugated fluorescent backbones. Capping on the polymer by activation can be done using appropriate functionalities, which results in the polymer being able to conjugate with the binding partner. Alternatively, the polymer may be activated for conjugation by attachment of appropriate functionalities outside the polymer backbone. The activated polymer may be conjugated to a binding partner. Any suitable binding partner (e.g., an antibody) may be used, followed by purification, for example, by using standard procedures. The functional group may be selected from the group consisting of amines, carbamates, carboxylic acids, carboxylic esters, maleimides, activated esters, N-hydroxysuccinimide groups, hydrazines, azides, alkynes, aldehydes, thiols and protected groups thereof for conjugation to a substrate or binding partner.

The polymer dye conjugate may comprise a fluorescent polymer having monomeric subunits including, but not limited to, dihydrophenanthrene (DHP), fluorene, and combinations thereof. In some embodiments, the polymer dye conjugate may comprise a polymer dye having the structure of formula III:

each a is independently selected from aromatic comonomers and heteroaromatic comonomers. Each a may be substituted with a functional group that will be conjugated to a binding partner.

Each optional M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene, and ethynylene groups, and is uniformly or randomly distributed along the polymer backbone. Each M may be independently selected from:

wherein each M may be substituted and terminated with a functional group selected from the group consisting of: amines, carbamates, carboxylic acids, carboxylic acid esters, maleimides, activated esters, N-hydroxysuccinimide groups, hydrazines, hydrazones, azides, alkynes, aldehydes, thiols, amides, sulfonamides, ethers, thioethers, thiocarbamates, hydroxy groups, iodoacetyl groups, hydrazino groups, ketones, phosphines, epoxides, ureas, thioureas, thioesters, imines, disulfides and protected groups thereof for binding to another substrate, acceptor dye, molecule or binding agent, and

Wherein each R ⁵ Independently selected from halogen, hydroxy, C ₁ -C ₁₂ Alkyl, C ₂ -C ₁₂ Olefins, 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, C ₂ -C ₁₈ (hetero) aryloxy, C ₂ -C ₁₈ (hetero) arylamino, carboxylic acid, carboxylic ester, (CH) ₂ ) _X’ (OCH ₂ —CH ₂ ) _y′ OCH ₃ And (CH) ₂ ) _x′ (OCH ₂ —CH ₂ ) _y′ OCF ₃ Wherein each x 'is independently an integer from 0 to 20 and each y' is independently an integer from 0 to 50; wherein the method comprises the steps of

Each R ¹ Independently an alkylammonium salt, an alkoxyammonium salt, an oligoether ammonium salt, an alkylsulfonate, an alkoxysulfonate, an oligoether sulfonate, an oligoether sulfonamide group, or the following moieties:

each R ² Independently is H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero) aryloxy, aryl, (hetero) arylamino, PEG groups, alkylammonium salts, alkoxyammonium salts, oligoether ammonium salts, alkylsulfonates, alkoxysulfonates, oligoether sulfonates, oligoether sulfonamide groups, or:

and is also provided with

each Z is independently selected from C, O and N;

Each subscript n is independently an integer of from 0 to 20.

The linker is represented as L in formula III. Each optional linker L may be an aryl or heteroaryl group, uniformly or randomly distributed along the polymer backbone, and may be substituted with one or more side chains terminated with a functional group selected from the group consisting of: amines, carbamates, carboxylic acids, carboxylic acid esters, maleimides, activated esters, N-hydroxysuccinimide groups, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof.

The polymer composites of the present disclosure further comprise a terminus represented in formula III, each G ¹ And G ² . The terminal end may be modified or unmodified. The terminal may each be independently selected from hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen substituted aryl, silyl, diazonium salt, trifluoromethanesulfonate, acetoxy, azide, sulfonate, phosphate, borate substituted aryl, borate, boric acid, optionally substituted Dihydrophenanthrene (DHP), optionally substituted fluorene, aryl or heteroaryl, substituted with one or more side chains terminated with a functional group selected from the group consisting of: amines, carbamates, carboxylic acids, carboxylic acid esters, maleimides, activated esters, N-hydroxysuccinimide groups, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof.

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

In some embodiments, the polymeric dye conjugate may have the structure of formula I:

wherein:

L ¹ 、L ² and L ³ Is a linker moiety;

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 Independently selected from unmodified polymer ends and modified polymer ends;

subscripts n and m are independently integers of from 1 to 10,000,

subscript p is an integer of from 0 to 10,000 and

the sum of subscripts n, m, and p ranges from 2 to 10,000;

subscript q is 1, 2, 3, or 4;

subscript r is 1, 2, 3, or 4;

subscript s is 0, 1, 2, or 3;

subscript t is 1 or 2

The sum of subscripts r and s ranges from 1 to 4; and is also provided with

A and B are randomly or non-randomly distributed in the conjugated polymer.

L ¹ May be a sulfonamide, sultam, disulfonamide, amide, phosphonamide, phosphonamic acid ester, phosphinamide, or secondary amine. Or 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, subscript s is 0 or 1 if subscript r is 1, and subscript s is 0 if subscript r is 2.

Each L ³ May be a covalent bond.

The conjugated polymer may have a structure according to formula II:

wherein:

L ^1a is a linker moiety; and is also provided with

R ¹ Selected from H and amine protecting groups.

Various linkers L as described herein may be utilized ^1a And L ² For the synthesis of polymers according to formula I and formula II. For example:

L ^1a can be selected from 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, 5-to 12-membered heterocyclylene, -NHC (O) L ^a -、-C(O)NHL ^a -、-C(O)L ^a -and combinations thereof;

L ² can be selected from 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, 5-to 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 Can be independently selected from C _1-8 Alkylene and 2 to 8 membered heteroalkylene; and is also provided with

R ¹ May be selected from H and amine protecting groups.

There is provided a polymer according to formula II, wherein:

L ^1a selected from covalent bonds, C _1-8 Alkylene, 2-to 8-membered heteroalkylene, -NHC (O) L ^a -、-C(O)NHL ^a -and-C (O) L ^a -，

L ² Selected from covalent bonds, C _1-8 An alkylene group; 2-to 8-membered heteroalkylene, -L ^b NHC(O)-、-L ^b C(O)NH-、-L ^b C(O)-、-C(O)NHL ^b -and-C (O) L ^b -；

L ^a And L ^b Independently selected from C _1-8 Alkylene and 2 to 8 membered heteroalkylene; and is also provided with

R ¹ Selected from H and amine protecting groups.

W may comprise one or more ethylene glycol monomers. Or W may comprise poly (ethylene glycol).

L ³ May be a trivalent arylalkyl moiety having: and the first L ¹ Part (or first L) ^1a Part) of the first connection point; and a second L ¹ Part (or second L) ^1a Part) of the first connection point; and a third point of attachment to the a monomer.

For example, the present disclosure provides conjugated polymers having two or more chromophores attached, as shown in formula VI:

wherein the method comprises the steps of

L ^1a As previously defined;

L ² as previously defined;

w is as previously defined;

L ^3a selected from covalent bonds, C _1-8 Alkylene, 2-to 8-membered heteroalkylene, -NHC (O) L ^a -、-C(O)NHL ^a -and-C (O) L ^a -；

L ^a Selected from C _1-8 Alkylene and 2 to 8 membered heteroalkylene; and the wavy line is the point of attachment to the a monomer.

Each a monomer in the polymer having the structure of formula I, II or III may be the same monomer. Each monomer in the 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-phenanthrenedione based monomer (e.g., a Dihydrophenanthrene (DHP) based monomer), a fluorene based monomer, or a fluorooxapine (fluoronoxepin) based monomer.

Monomer a in the polymer having the structure of formula I, II or III may be a DHP-based monomer, for example:

wherein:

each X is independently C or Si;

each Y is independently CR ¹ R ² Or SiR ¹ R ² ；

Each R ¹ Independently an alkylammonium salt, an alkoxyammonium salt, an oligoether ammonium salt, an alkyl sulfonate, an alkoxy sulfonate, an oligoether sulfonate, a sulfonamide oligoether, or a combination thereofThe method comprises the following steps:

each R ² Independently is H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero) aryloxy, aryl, (hetero) arylamino, PEG groups, alkylammonium salts, alkoxyammonium salts, oligoether ammonium salts, alkylammonium sulfonates, alkoxyalkoxysulfonates, oligoether sulfonates, oligoamide-based polyethers, or:

each Z is independently selected from C, O and N;

Each subscript n is independently an integer of from 0 to 20.

R ¹ There may be a structure shown below, wherein Q is NH:

each R ³ Independently selected from H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero) aryloxy, aryl, (hetero) arylamino, and PEG groups; and each Z is independently selected from C, O and N.

The DHP-based monomer may have the following structure:

wherein:

each subscript f is independently an integer of from 0 to 50;

each subscript n is independently an integer of from 0 to 20;

each R ² Independently is H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero) aryloxy, aryl, (hetero) arylamino, PEG groups, alkylammonium salts, alkoxyammonium salts, ammonium oligoether salts, alkyl sulfonates, alkoxy sulfonates, oligoether sulfonates, sulfonamide oligoethers, or:

each R ⁵ H, C independently 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 A heteroaromatic amino group; and is also provided with

Each Z is independently selected from C, O and N.

The DHP monomer may have the following structure:

wherein:

each subscript f is independently an integer of from 0 to 50;

each subscript n is independently an integer of from 0 to 20;

Each Z is independently selected from C, O and N.

The monomer a in the polymer having the structure of formula I, II or III may be a fluorene-based monomer, for example:

therein X, Z, R ¹ 、R ² 、R ⁵ Subscript n, subscript f are as defined herein.

R ¹ Radicals and R ² Groups such as alkylammonium salts, alkoxyammonium salts, ammonium oligoether salts, alkyl sulfonates, alkoxy sulfonates, oligoether sulfonates, sulfonamide oligoethers or moieties having the following structure may impart solubility in water/buffer:

in some embodiments, for example, the polymer is soluble at the following levels: more than 10mg/mL, more than 15mg/mL, more than 20mg/mL, more than 25mg/mL, more than 30mg/mL, more than 35mg/mL, more than 40mg/mL, more than 45mg/mL, more than 50mg/mL, more than 60mg/mL, more than 70mg/mL, more than 80mg/mL, more than 90mg/mL, or more than 100mg/mL.

Monomer a also includes bridging monomers. For example, the bridging monomers of the present invention include:

therein, X, Y, R ² And R is ⁵ As defined previously.

The monomer a in the polymer having the structure of formula I, II or III may be an oxazepine-based monomer (e.g., a fluorenooxapine-based monomer), for example:

Therein X, R ¹ And R is ² As defined herein.

Tandem polymeric dyes

The polymer may have an acceptor dye attached to the backbone, which will provide monitoring of the emission of the acceptor dye attached to the backbone by an energy transfer linkage. Acceptor dyes that may be used in tandem polymer dyes include, for example, FITC, CY3B, cy, aleXa 488, texas red, CY5, CY7, aleXa 750, and 800CW. For example, the acceptor dye may be attached to the polymer via a linker L:

as described in U.S. published application No.2020/0190253, which is incorporated herein by reference in its entirety, the acceptor dye may also be directly attached to monomer a as in group E in the structures of figures I or II above. SuperNova ^TM Tandem dye SuperNova ^TM v605 and SuperNova ^TM v786 (Beckman Coulter, inc.) is a tandem polymer dye derived from the core SuperNova ^TM v428. Both SuperNova v605 and SuperNova v786 have the same absorption characteristics, with the excitation maximum at 414 nm. The best detection was achieved using 610/20 and 780/60nm bandpass filters of the flow cytometer with emission peaks at 605nm and 786nm, respectively.

Conjugate dyes

The polymeric dye can be conjugated with a different specific binding partner (e.g., a target analyte-specific antibody) to synthesize a binding partner-dye conjugate, such as CD19-SN v428, CD20-SN v605, and the like.

The polymer dye and polymer dye conjugate may be formulated with an aqueous buffer. Any suitable aqueous buffer may be used, for example an isotonic aqueous buffer, for example PBS buffer. The aqueous buffer may contain additives. For example, as described herein, the aqueous buffer may comprise BSA, sodium azide, a nonionic surfactant (e.g., PF-68), and a zwitterionic surfactant (e.g., empigen) Or an anionic surfactant (e.g., NLS). BSA helps to stabilize the conjugate, sodium azide prevents any microbial contamination, and surfactants (e.g., empigen +.>) Non-specific binding on monocytes and granulocytes is significantly reduced or eliminated. BSA may be present in a range of 0 to 3mg/mL, 0.5 to 2.5mg/mL, or about 2 mg/mL. Sodium azide may be present in the range of 0 to 0.05%, 0.05% to 0.03%, or about 0.01% (w/v).

Binding partners

As used herein, a "binding partner" refers to any molecule or molecular complex capable of specifically binding to a target analyte. The binding partner may be, for example, a protein (e.g., an antibody or antigen-binding antibody fragment), a small organic molecule, a carbohydrate (e.g., a polysaccharide), an oligonucleotide, a polynucleotide, a lipid, an affinity ligand, an aptamer, or the like. In some embodiments, the binding partner is an antibody or fragment thereof. In the context of the present disclosure, specific binding 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 particular binding partner preferentially binds to a particular protein or isoform of a particular protein, but not to other proteins or other isoforms present in the sample in significant amounts.

In some cases, the antibodies include intravenous immunoglobulins (intravenous immunoglobulin, IVIG) and/or antibodies from IVIG (e.g., enriched from IVIG, purified from IVIG, e.g., affinity purified from IVIG). IVIG is a blood product comprising IgG (immunoglobulin G) (immunoglobulin G) pooled from the plasma (e.g., in some cases free of any other proteins) of many (e.g., sometimes over 1,000 to 60,000) normal and healthy blood donors. IVIG is commercially available. Aspects of IVIG are described, for example, in U.S. patent application No.2010/0150942;2004/0101909;2013/0177574;2013/0108619; and 2013/0011388, which is incorporated herein by reference.

When the binding partners are antibodies, they may be monoclonal or polyclonal antibodies. The term "antibody" as used herein refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin (Ig) molecule, e.g., that specifically binds to an antigen in a target analyte. Such antibodies include, but are not limited to, polyclonal, monoclonal, monospecific polyclonal antibodies, antibody mimics, chimeric antibodies, single chain antibodies, fab 'and F (ab') ₂ Fragment, fv and Fab expression libraries. In some cases, the antibody is a monoclonal antibody defining subclasses (e.g., igG1, igG2, igG3, or IgG4, igA, igD, igE, igG2a, igG2b, igG3, and IgM). If 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, the monoclonal antibody is humanized. Antibody fragments may comprise, for example, fab, scFv, F (ab') ₂ And Fab' molecules. Antibody derivatives include antibodies or fragments thereof with additions or substitutions, such as chimeric antibodies. Antibodies may be derived from human or animal sources, from hybridomas, by recombinant methods or any other means known in the art.

Binding partners other than antibodies or target analyte-specific antibody fragments or derivatives may also be used in the present systems and methods. For example, the binding partner may be a nucleic acid or nucleic acid analogue, such as an oligonucleotide or PNA probe. In one embodiment, an aptamer may be used as a specific binding partner. Aptamers are single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can bind with high affinity and specificity to pre-selected targets, including proteins and peptides. Other binding partners that bind to the target analyte to form acceptor-ligand pairs, enzyme-substrate pairs, enzyme-inhibitor pairs, and enzyme-cofactor pairs may also be used. Some specific examples of such binding partner pairs include carbohydrates and lectins, biotin and avidin or streptavidin, folic acid and folic acid binding proteins, vitamin B12 and intrinsic factors, protein a and immunoglobulins, and protein G and immunoglobulins. Binding partners that form covalent bonds with the target analyte are also included.

Conjugation

The polymer dye conjugate may comprise any known polymer dye conjugated to a binding partner using techniques known to those skilled in the art. In some embodiments, the polymer dye may be conjugated to the binding partner to form a polymer dye conjugate using a direct modification of the core polymer described in U.S. published application No.2020/0190253, which is incorporated herein by reference in its entirety.

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

SuperNova v428 (Beckman Coulter) is a bright polymer dye that can be activated with an amine for tandem dyes and then with maleimide for tandem conjugates.

Target analytes

The present disclosure also relates to methods 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 some analytical procedure. The present disclosure is directed to detecting the presence, and in some cases the amount, of a particular target analyte. The term "target analyte" refers to a target molecule, such as peptides, proteins, polynucleotides, organic molecules, sugars and other carbohydrates, lipids and small molecules, comprising a target antigen to be detected in a biological sample. An important aspect of the present disclosure is that the target analyte is contained in a liquid sample and is accessible, or accessible at some point, to bind a target analyte-specific binding partner of the present invention. The target analyte may be present in a biological sample, such as a blood sample, a cell line development sample, a tissue culture sample, and the like.

The target analyte may be, for example, a nucleic acid (DNA, RNA, mRNA, tRNA or rRNA), peptide, polypeptide, protein, lipid, ion, monosaccharide, oligosaccharide, polysaccharide, lipoprotein, glycoprotein, glycolipid, or fragment thereof. The target analyte may be a protein, and may be, for example, structural microfilaments, microtubule and intermediate filament proteins, organelle specific markers, proteasomes, transmembrane proteins, surface receptors, nucleoporins, protein/peptide translocases, protein folding partners, signaling scaffolds, ion channels, and the like. 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 binding and modifying proteins and/or RNA binding and modifying proteins, nuclear import and export receptors, apoptosis or survival modulators, and the like.

The target analyte may be present and accessible on the cell surface. Illustrative examples of useful analytes include, but are not limited to, the following: 1) Specific cell surface macromolecules and antigens (including hormones, protein complexes and molecules recognized by cellular receptors) and 2) permeabilizing cellular proteins, DNA or RNA in cells, including abnormal DNA or RNA sequences or abnormal amounts of certain messenger RNAs. Detection of these analytes may be particularly useful where they are contained in rare cells and/or where they are identifiers of rare cells, such as where they are present in the early stages of a variety of cancers.

In some examples, the target analyte may be CD2, CD3, CD4, CD8, CD10, CD11c, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD27, CD38, CD45RA, CD56, CD62L, CD, CD95, CD103, HLA-DR, IFN-gamma, TNF-alpha, or ZAP-70, or other target analyte of interest.

Biological sample

Some non-limiting examples of biological samples include blood, serum, plasma, urine, semen, milk, sputum, mucus, cheek swab, vaginal swab, rectal swab, aspirate, needle biopsy, such as tissue sections obtained by surgery or autopsy, plasma, serum, spinal fluid, lymph fluid, skin, respiratory, intestinal and genitourinary secretions, tears, saliva, tumors, organs, samples of in vitro cell culture constituents including, but not limited to, conditioned medium produced by cell growth in cell culture medium, putative virus infected cells, recombinant cells and cell components.

The sample in the methods of the present disclosure may be, for example, blood. The blood sample may be whole blood. Whole blood may be obtained from a subject using standard clinical procedures. The sample may be a subset of one or more cells (e.g., erythrocytes, leukocytes, 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) of whole blood. The sample may be from a cell culture. The sample may naturally contain the target analyte or the sample may be prepared in whole or in part by synthetic means.

Object(s)

The subject may be a human (e.g., a patient suffering from or suspected of suffering from a disease), a commercially important food chain mammal (including, e.g., cattle, steer, pigs, goats, sheep, birds, fish, or horses). Samples may also be obtained from domestic pets or companion animals including, for example, dogs, cats, rabbits, birds, or ferrets. The subject may be a laboratory animal, such as a monkey, mouse, rat, rabbit or guinea pig, for use as an animal model of a disease or for drug screening. The subject may be a exotic animal, such as a zoo animal or a wild animal, such as elephant, antelope, zebra, bison, giraffe, lion, tiger, leopard, gorilla, whale, dolphin, shark, or reptile.

Reaction vessel

The reaction vessel disclosed herein may be any vessel in which a reaction between a binding partner or its polymer dye conjugate and a target analyte can occur. For example, the reaction vessel may be a tube, a plate, a well of a microtiter plate, a chamber, and a slide. In a preferred embodiment, the reaction vessel has a cover or cap so that the binding reaction can occur in a closed environment.

Substrate

The reaction vessel comprises one or more substrates. The substrate may be any suitable surface including, but not limited to, plastic, nitrocellulose, cellulose acetate, quartz, and glass. Some non-limiting examples of plastics may include polystyrene, polypropylene, cyclic olefins, and polycarbonate. In some embodiments, the substrate is a film. The substrate may be an inner surface of a reaction vessel body, such as a well of a plastic tube or a microtiter plate. The substrate may also be a bead. In some embodiments, at least one substrate (e.g., a membrane) that receives the labeled binding partner is bonded to an inner surface of the reaction vessel body. In some embodiments, the film substrate is a sheet or roll, which makes it easier to deposit the solution and easier to dry. In some embodiments, the membrane may be cut to separate individual dried reactant spots. In some embodiments, the cut film simply falls into the reaction vessel. In some preferred embodiments, the cut film is bonded to the surface of the reaction vessel such that the spot does not escape from the vessel when liquid is pipetted into or out of the reaction vessel.

Liquid sample

The reaction vessel is configured to receive a liquid sample. The liquid sample used in the present invention typically comprises the target analyte obtained as the primary aqueous medium or dispersed in the primary aqueous medium.

The sample may be, for example, a biological sample, such as blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirate (or any cells obtained from bone marrow), urine (lavage), serum, plasma, saliva, cerebrospinal fluid, lymph, urine, amniotic fluid, interstitial fluid, stool, mucus, milk, semen, cheek swab, nasopharyngeal swab, vaginal swab, rectal swab, aspirate, needle biopsy, a tissue slice or tissue (e.g., a tumor sample, a decomposed tissue, a decomposed solid tumor) sample obtained, for example, by surgery or autopsy. The sample may be a blood sample. The blood sample may be a whole blood sample. Whole blood may be obtained from a subject using standard clinical procedures. The sample may be a subset of one or more cells of whole blood (e.g., erythrocytes, leukocytes, 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). Samples may be derived from cell cultures, in vitro cell culture constituents (including but not limited to conditioned media produced by the growth of cells in cell culture media, putative virus infected cells, recombinant cells, and cell components).

The sample may be of any source of biological material and may comprise proteins, carbohydrates and/or polynucleotides obtainable directly or indirectly from a living organism. The sample may comprise, for example, cells, tissues or fluids, as well as deposits left by organisms, including viruses, mycoplasma and fossils. The sample may comprise a target analyte. The target analyte may be naturally present in the biological sample or may be prepared wholly or partially synthetically.

Labeled binding partners

The dye may be conjugated to the binding partner by a variety of linking chemistries between the binding partner and the reactive pair in the label. Reactive pairs may include, but are not limited to, maleimide/thiol, succinimidyl ester (NHS ester)/amine, azide chemistry, carboxyl/EDC (1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride)/amine, amine/sulfo-SMCC (sulfosuccinimidyl 4- [ N-maleimidoethyl ] cyclohexane-1-carboxylate)/thiol, and amine/BMPH (N- [ -maleimidopropionic acid ] hydrazide. TFA)/thiol. Methods for performing conjugation are well known in the art. Commercially available kits for performing conjugation are also readily available, such as kits from Innova biosciences (Cambridge, UK), novus Biologicals (Littleton, colo.), thermo Fisher Scientific (Waltham, mass.).

Dry or liquid polymer dye conjugates can be used in the methods and compositions. The dried polymer dye conjugate may be prepared using any technique known in the art. The techniques may be as described in US 2019/0202022882, which is incorporated herein by reference.

The polymer dye conjugates can be used in compositions according to the present disclosure, which can be used directly to stain blood and analyze in a flow cytometer.

Measurement system

Assay systems are known that use binding partners and fluorescent labels to quantify the bound molecules. Some examples of such systems include flow cytometry, scanning cytometry, imaging cytometry, fluorescence microscopy, and confocal fluorescence microscopy.

Flow cytometry is used to detect fluorescence. Many devices suitable for this purpose are available and known to those skilled in the art. Some examples include BCI Navios, gallios, aquios and CytoFLEX ^TM Flow cytometer.

The assay may be an immunoassay. Some examples of immunoassays that can be used in the present disclosure include, but are not limited to, fluorescent luminescence assays (fluoroluminescence assay, FLA), and the like. The assay may also be performed on a protein array.

When the binding partner is an antibody, an antibody or multi-antibody sandwich assay may also be used. Sandwich assays refer to the use of successive recognition events to establish layers of multiple binding partners and reporter elements to indicate the presence of a particular analyte. Some examples of sandwich assays are disclosed in U.S. patent No.4,486,530 and references mentioned therein.

A light source is applied to the sample, which light source can excite the polymer and detect light emitted from the conjugated polymer complex. In a typical assay, the fluorescent polymer dye conjugates used in the present invention can be excited by light having wavelengths of about 395nm and about 415 nm. The emitted light is typically about 415nm and about 475nm. Alternatively, the excitation light may have a wavelength of about 340nm to about 370nm, and the emission light is about 390nm to about 420nm.

Application of

The compositions according to the present disclosure may comprise a single color, i.e., a single polymer dye conjugate, such as a single SN polymer dye conjugate. For example, a biological sample can be stained with SN conjugates to monitor or determine a particular cell population, depending on the antibody conjugated to the polymer dye.

In some embodiments, compositions according to the present disclosure may comprise a single color polymer dye conjugate as well as conventional non-polymer dye conjugates. For example, SN conjugates can be used with non-polymeric dye conjugates such as CD4-FITC, CD7-PE, CD25-ECD, CD56-PC5.5, and the like in one group to determine cell subsets in human whole blood samples by flow cytometry.

In some embodiments, one or more compositions according to the present disclosure may be contacted with a biological sample (e.g., a blood sample). For example, biological samples can be stained with compositions comprising a plurality of SN conjugates to monitor or determine specific cell populations based on antibodies conjugated to polymer dyes. In some embodiments, 2 or more, 3 or more, or 4 compositions according to the present disclosure may be contacted with a biological sample. In some embodiments, compositions comprising multiple polymer dye conjugate compositions may also comprise non-polymer dye conjugates in one group, such as CD4-FITC, CD7-PE, CD25-ECD, CD56-PC5.5, and the like, to determine cell subsets in a human whole blood sample by flow cytometry.

Examples

The invention may be better understood by reference to the following examples which are provided by way of illustration. The invention is not limited to the examples given herein.

Example 1: preparation of DHP Polymer complexes

Method 1: in a round bottom flask, as described in WO 2017/180998, dibromodhp monomer and diboron DHP monomer (1:1) to (DMF-water) mixture were taken and purged with nitrogen for 10 minutes. About 20 equivalents of CsF and 10% Pd (OAc) under nitrogen ₂ Mix and heat at 80 degrees celsius. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. Thereafter, a capping agent (selected from G1) containing suitable functional groups was added to the reaction mixture, and a second capping agent (selected from G2) was added after 3 hours. 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. Washing with 20% ethanol was performed until the absorption of the filtrate was reduced.

Method 2: alternatively, the polymerization may be accomplished by self-polymerizing bromoborates of the DHP molecule. In a round bottom flask, DHP bromoborate was taken into a (DMF-water) mixture and purged with nitrogen for 10 min. About 10 equivalents of CsF and 5% Pd (OAc) under nitrogen ₂ Mix and heat at 80 degrees celsius. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. Thereafter, a capping agent (selected from G1) containing suitable functional groups was added to the reaction mixture, and a second capping agent (selected from G2) was added after 3 hours. 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. Washing with 20% ethanol was performed until the absorption of the filtrate was reduced.

Method 3: taking dibromodihydrophenanthrene and diboron in a round bottom flaskBoth hydronaphthalene monomers (1:1) and were dissolved in a solution containing 10 equivalents of K ₂ CO ₃ And 3% Pd (PPh) ₃ ) ₄ THE THE-water (4:1) mixture. The reaction mixture was placed on a Schlenk line and degassed with three freeze-pump-thaw cycles and then heated to 80 degrees celsius under nitrogen with vigorous stirring for 18 hours. Thereafter, a capping agent (selected from G1) containing a suitable functional group was added to the reaction mixture through a cannula under an excess of nitrogen, and a second capping agent (selected from G2) was added after 3 hours. 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. Washing with 20% ethanol was performed until the absorption of the filtrate was reduced.

Method 4: alternatively, the polymerization may be accomplished by self-polymerizing bromoborates of dihydrophenanthrene molecules. In a round bottom flask, dihydrophenanthrene bromoborate was taken and dissolved in a flask containing 10 equivalents of K ₂ CO ₃ And 3% Pd (PPh) ₃ ) ₄ In a THF-water (4:1) mixture. The reaction mixture was placed on a Schlenk line and degassed with three freeze-pump-thaw cycles and then heated to 80 degrees celsius under nitrogen with vigorous stirring for 18 hours. Thereafter, a capping agent (selected from G1) containing a suitable functional group was added to the reaction mixture through a cannula under an excess of nitrogen, and a second capping agent (selected from G2) was added after 3 hours. 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. Washing with 20% ethanol was performed until the absorption of the filtrate was reduced.

Example 2: preparation of fluorene-DHP copolymer complexes

Method 1: in a round bottom flask, dibromoDHP and diboron fluorene monomers (1:1) to (DMF-water) mixture were taken and purged with nitrogen for 10 minutes. About 20 equivalents of CsF and 10% under nitrogenPd(OAc) ₂ Mix and heat at 80 degrees celsius. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. Thereafter, a capping agent (selected from G1) containing suitable functional groups was added to the reaction mixture, and a second capping agent (selected from G2) was added after 3 hours. 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. Washing with 20% ethanol was performed until the absorption of the filtrate was reduced.

Method 2: in a round bottom flask, both dibromofluorene and diboron DHP monomers (1:1) were taken into a (DMF-water) mixture and purged with nitrogen for 10 minutes. About 20 equivalents of CsF and 10% Pd (OAc) under nitrogen ₂ Mix and heat at 80 degrees celsius. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. Thereafter, a capping agent (selected from G1) containing suitable functional groups was added to the reaction mixture, and a second capping agent (selected from G2) was added after 3 hours. 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. Washing with 20% ethanol was performed until the absorption of the filtrate was reduced.

Method 3: in a round bottom flask, both dibromophenanthrene and diboron fluorene monomers (1:1) were taken and dissolved in a flask containing 10 equivalents K ₂ CO ₃ And 3% Pd (PPh) ₃ ) ₄ Is added to the water (4:1) mixture. The reaction mixture was placed on a Schlenk line and degassed with three freeze-pump-thaw cycles and then heated to 80 degrees celsius under nitrogen with vigorous stirring for 18 hours. Thereafter, a capping agent (selected from G1) containing a suitable functional group was added to the reaction mixture through a cannula under an excess of nitrogen, and a second capping agent (selected from G2) was added after 3 hours. 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. Washing with 20% ethanol was performed until the absorption of the filtrate was reduced.

Method 4: in a round bottom flask, takeDibromofluorene and diboron dihydrophenanthrene monomers (1:1) and dissolved in a solvent containing 10 equivalents K ₂ CO ₃ And 3% Pd (PPh) ₃ ) ₄ In a THF-water (4:1) mixture. The reaction mixture was placed on a Schlenk line and degassed with three freeze-pump-thaw cycles and then heated to 80 degrees celsius under nitrogen with vigorous stirring for 18 hours. Thereafter, a capping agent (selected from G1) containing suitable functional groups was added to the reaction mixture through a cannula under excess nitrogen pressure, and a second capping agent (selected from G2) was added after 3 hours. 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. Washing with 20% ethanol was performed until the absorption of the filtrate was reduced.

Example 3: comparison of fluorescence emission spectra

Fluorescence emission spectra of fluorene (Fl-Fl), dihydrophenanthrene (DHP-DHP) and fluorene-DHP (DHP-Fl) polymers were compared. As shown in fig. 1A, after excitation at 405nm, the DHP-containing polymer showed a significant difference in fluorescence maximum at 426 to 428nm, while the fluorene-based polymer showed a maximum at 421 nm.

Example 4: comparison of absorption spectra

Absorption spectra of both fluorene (Fl-Fl) polymer and dihydrophenanthrene (DHP-DHP) polymer were measured. As shown in FIG. 1B, the DHP-DHP polymer (black curve) exhibits a maximum lambda value (λmax) at 390 and 410nm, while the Fl-Fl (gray curve) polymer exhibits a maximum lambda value (λmax) at about 400 nm. Samples were measured at different concentrations.

Example 5: polymeric dye Properties

The polymeric dyes of the present disclosure were found to have the following specific physical and chemical properties when conjugated to antibodies: absorption, fluorescence, brightness, molecular weight, polydispersity, dye to protein ratio, and the like. Preferred ranges for these parameters are shown in table 1A.

TABLE 1A Polymer dye Properties

Excitation and emission spectra of the tandem polymers were measured. Excitation was performed at the polymer maximum (405 nm) and emission was observed from a variety of acceptor dyes on the backbone.

Example 6: experiment of unconjugated dye

With and without EmpigenIn the case of (a) blood samples were stained with unconjugated polymeric dye SN v605 (no antibody) and analyzed in a flow cytometer. As shown in the lower right of FIG. 2, EMPIGEN->The presence of (c) shows that the non-specific interaction of fluorescent polymers conjugated to the binding partner with leukocytes in the blood is effectively reduced. In fig. 2, fluorescent polymer dye SN v605 without antibody was used to stain blood samples and analyzed in a flow cytometer. The lower left of FIG. 2 shows that there is no Empigen +.>In the presence of a polymeric dye that non-specifically binds to monocytes/granulocytes. While not wishing to be bound by any particular theory, it is believed that the polymer may adsorb on the cell surface of monocytes and granulocytes. When EMPIGEN->When the cell surface is EMPIGEN->The molecules are blocked and the non-specific binding of the polymer to monocytes and granulocytes is greatly reduced.

At the time of adding EMPIGENIn the case of the polymer, the stippled plot looks like an undyed sample of the plot on fig. 2 that does not contain any polymer. The diffusion of granulocyte population was comparable to that of unstained tubes, indicating that when EMPIGEN +. >When added to the polymeric dye, the non-specific interactions of the polymeric dye with monocytes and granulocytes are greatly reduced.

Example 7: experiments with conjugated dye SN 605-CD20

EMPIGENConjugates with those described herein (e.g., SN 605-CD20, SN786-CD103, and SN428 conjugates), bovine serum albumin (BSA; 2 mg/mL), sodium azide (0.1%) and pluronic F-68 (polyethylene oxide-polypropylene oxide-polyethylene oxide nonionic triblock copolymer) were formulated at a dose of 0.12%/10. Mu.L of the conjugate.

CD20 is a B-cell marker expressed during pre-B lymphocyte development, persists in B lymphocyte expression, and loses its expression as plasma cells differentiate. CD20 is not expressed on other leukocyte populations including monocytes, granulocytes and NK cells. FIG. 3 shows that in the absence of EMPIGENAnd the presence of EMPIGEN->SN 605-CD20 conjugate.

In using EMPIGENIn the mean time, the percentage of nonspecifically bound granulocytes is reduced (see "P2" in the dot pattern). Furthermore, the functional aspects of the conjugates were not altered (see "P1" gating in the dot plots) as the percentage of positive populations was similar in both cases.

To confirm EMPIGENIs in the presence and absence of EMPIGEN->Two batches of SN 605-CD20 conjugate were tested. The Mean Fluorescence Intensity (MFI) was compared to the autofluorescence (median fluorescence intensity of negative population (MDFI)) from monocytes from unstained samples. The results shown in FIG. 4 demonstrate that in the presence of surfactant, the nonspecific interactions on monocytes were reduced to 75% and 67% for the Lot-1 and Lot-2 SN605 CD20 conjugates, respectively. Similar effects (reduction of non-specific binding) were also observed in granulocytes, but the percent reduction was not significant (13.7% and 17.7% for Lot-1 and Lot-2, respectively, FIG. 5).

Example 8: experiments with conjugated dye SN 786-CD103

CD103 conjugates were tested on the cell line (MOLT 16) because they are not normally expressed in normal whole blood. Since there is no expression of CD103 in normal blood, there should be no positive signal. However, SN786 CD103 conjugates also tended to bind to whole blood due to non-specific interactions. Adding EMPIGEN to the preparationHelping to inhibit such non-specific interactions. This is shown in fig. 6.

In the presence and absence of EMPIGEN Two batches of SN 786-CD103 conjugate were tested and autofluorescence of monocytes and granulocyte populations (negative population MDFI) was compared against unstained samples. As shown in figure 7, for SUPERNOVA ^TM Lot-1 and Lot-2 of the conjugates, signals from nonspecific binding of the conjugates to monocytes were reduced by 149.3% and 202.1%, respectively. In granulocyte populationsThe same effect (reduced non-specific binding) was also observed, as shown in FIG. 8, with 150.8% and 253% reduction for Lot-1 and Lot-2, respectively.

Example 9: effect of zwitterionic surfactants on reduction of monocyte background with SN 428 conjugates

Evaluation of zwitterionic surfactant EMPIGENReducing the efficacy of non-specific binding to monocytes. Other populations, i.e., lymphocytes and granulocytes, were also studied to evaluate the detergent's no effect on MFI and percent of cells.

The experimental conditions are generally as follows:

CD19-SN 428 (lot number D19-094, polymer lot number RDS-042919 (82.7 kD), 1 dose (0.5. Mu.g/test).

CD22-SN 428 (lot D19-109, polymer lot WX-20190624 (86.4 kD), 1 dose (0.5. Mu.g/test).

CD25-SN 428 (lot number D19-107, polymer lot number RDS-062419 (72.8 kD), 1 dose (0.5. Mu.g/test).

The commercial doses were 1X for CD19, CD22 and CD25-BV 421 from Becton Dickinson.

Preparation of three doses of EMPIGEN in conjugation final formulation0.06%, 0.12% and 0.2%.

10 μl sample in 100 μl whole blood was added.

2 donors lysed with VersaLyse (lysis solution for lysing erythrocytes, beckman Coulter, inc.) +fix were tested, washed once.

The o Navios flow cytometer was collected as FL 9.

The required x number of test tubes is prepared, where "x number of test tubes" depends on the performance of the test. A calculated volume of conjugated antibody (at the required dose) was added to each tube. Whole blood (100. Mu.L) was added to each tube. The tube was gently vortexed for 15 seconds and incubated at 18 to 25℃for 15 to 20 minutes at room temperature protected from light. Versalysise and IOTest3 fixative mixture (2mL Versalyse Ref.A09777+50. Mu.L IOTest3 fixative 10X Ref. A07800) was added to the tube. The tube was immediately vortexed for 1 second and incubated at room temperature (18-25 ℃) for 10 minutes in the absence of light. The tube was centrifuged at 300g for 5 min at room temperature, the supernatant removed by aspiration, and the cell pellet resuspended using 3mL of PBS 1X. The tube was centrifuged again at 300g for 5 min at room temperature (18-25 ℃) and the supernatant removed by aspiration and the cell pellet resuspended using 0.5ml of PBS 1X or PBS 1X formaldehyde 0.1% (obtainable by dilution of 1ml of PBS 1X+12.5. Mu.l of IOTest3 fixative 10X).

In cytometry, compensation is a mathematical correction of signal overlap between different fluorescent dye emission spectrum channels. Thus, the correction factor is used to eliminate signal leakage into other undesired channels. Manual compensation was performed to assess conjugate performance.

Table 1B shows the use of CD19, CD22 and CD25 SuperNova ^TM Raw data for v428 conjugate, relatively EMPIGEN free ^TM Normalized data under conditions and final% reduction in background staining of monocytes obtained on 2 donors. The data show that in the presence of Empigen compared to conditions without Empigen at maximum monocyte backgroundIn the case of (2), the nonspecific background monocyte binding was significantly reduced by 52% to 73%.

TABLE 1B background reduction analysis of Empigen on monocytes

MFI = mean fluorescence intensity

w/o = none

w/= have

Table 2 shows EmpigenReduced granulocyte backgroundIs used. Compared to the condition without EMPIGEN at maximum granulocyte background, a 4% to 21% decrease in granulocyte background was found.

And II. Analysis of granulocyte Empigen background reduction

Table 3 showsEffect on positive lymphocyte populations: in contrast to the condition without the EMPIGEN,does not induce a significant change in positive signals on lymphocytes.

TABLE 3 analysis of Empigen effect on positive lymphocytes

The data in tables 1 to 3 are summarized in figures 11 to 15.

Table 4 describes additional experiments showing the percent background reduction on monocytes and granulocytes.

TABLE 4 percent background reduction on monocytes and granulocytes

Example 10: EMPIGENEffect on sample cell integrity

EMPIGENAre surfactants and can therefore cause cell membrane permeabilization, leading to cell death. From the studies described herein it can be concluded that EMPIGEN +.for use with polymer dye conjugates>Does not induce whole blood cell permeabilization or death and does not affect the performance of the conjugate.

Micelle concentration in the samples and during staining were studied to ensure that the critical micelle concentration (critical micellar concentration, CMC) was not exceeded. Referring to table 5, the evaluation of CMC in the conjugate formulation and the evaluation of CMC during staining are shown. CMC in the conjugate formulation in 100 μl whole blood and CMC during staining were studied. Experiments were performed to evaluate the addition of EMPIGENEffect on whole blood cell integrity and peripheral blood mononuclear cells (peripheral blood mononuclear cell, PMBC).

TABLE 5 Empigen in conjugate formulations Concentration and Empigen +.>Concentration of

The percentage of dead cells in the whole blood sample with 7-AAD was evaluated. 7-AAD is a DNA marker, when cell membrane permeabilization, staining is positive. Testing in Whole blood with four donors with 7-ADD in the absence of EMPIGENIn the case of (negative control) 0.06%, 0.12% and 0.2% EMPIGEN +.>In the following, to evaluate the percentage of dead cells under each condition. The purpose of the experiment was to verify if the percentage of dead cells was not determined by EMPIGEN +.>The concentration is increased.

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

Staining protocol for 7-AAD:

100. Mu.L of whole blood +10. Mu.L of CD19-SN v428+/-Empigen were incubated for 20 min.

The omicron is cracked by Versalysine and washed once,

o resuspended in 500. Mu.L PBS 1X

Add 20. Mu.L 7-AAD (Ref B88526), incubate for 15 to 20 minutes

Collecting by an o Navios flow cytometer: FL4 for 7-AAD, FL9 for CD19-SNv428

The data is shown in fig. 16.

In summary, EMPIGEN has been demonstratedEffectively reducing non-specific interactions of the polymer dye conjugates described herein (SN 605-CD20, SN786-CD103, SN428-CD25, SN428-CD19, and SN428-CD 22). When five specificities of the conjugates were tested, EMPIGEN +. >Effectively reducing non-specific background binding to monocytes and granulocyte populations. EMPIGEN->Is not donor dependent. When the conjugate is conjugated with BV786-CD10There was a clear difference in the reduction of nonspecific pullout of monocytes when compared to 3 and BV 421. Besides its properties, EMPIGEN->The presence of the conjugate with the polymer dye does not induce permeabilization of whole blood cell membranes and does not induce death of whole blood cells in the composition at concentrations at least as high as 0.2%.

Example 11: action of nonionic surfactants

The non-ionic surfactants Tween-20, tergitol, NP-40 and Pluronic F-68 (PF-68) are additional detergents/surfactants that were tested to remove non-specific binding of the conjugates described herein on monocytes. FIG. 9 shows the inefficiency of Tween-20 and PF-68 in avoiding non-specific binding of conjugates on monocytes.

Example 12: function of protein blocking agent

Non-specific interaction problems on monocytes were also observed with conventional tandem dyes (e.g., PC5, PC5.5, PC7, AA700 available from Beckman Coulter, inc.). To overcome this problem, it is believed that the known protein blockers BSA-ox (oxidized BSA) and BSA-Cy5-ox (oxidized Cy 5-BSA) prevent non-specific binding. However, none of BSA, BSA-ox and BSA-Cy5-ox was found to be effective in controlling the nonspecific interactions of the polymer dye conjugates with granulocytes/monocytes. See fig. 10.

Example 13: effect of anionic surfactants on non-specific binding

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

NLS is an anionic surfactant with CMC of 14.57mM (30 ℃). NLS sodium was evaluated to determine the effective concentration for preventing non-specific binding of the polymer dye conjugate (SN v605-CD 20) on monocytes and granulocytes.

In this example, NLS was formulated with SN v605 CD20 conjugate at different concentrations (0.16%, 0.08%, 0.04% and 0.02% w/v) in the presence of BSA and sodium azide, and peripheral blood samples were stained. The sample is stained followed by flow cytometry.

Dot plots of blood samples in the absence and presence of conjugate (with/without Empigen/NLS at different concentrations) are shown in fig. 17A-E. A dot pattern of a peripheral blood sample without a monochromatic conjugate is shown in fig. 17A, demonstrating the absence of populations in the cd20+ gate.

In the presence of BSA, sodium azide and zwitterionic surfactant EmpigenA positive control dotted plot of peripheral blood samples in the presence of CD20-SN v605 single color conjugate in the buffer composition as additive is shown in fig. 17B. When compared to the negative control stippling (fig. 17C), the% populations in gating "Mons non-specific binding" (0.64%) and "Grans non-specific binding" (0.68%) were each significantly reduced, indicating Empigen- >Effective in preventing nonspecific binding to monocytes and granulocytes.

A negative control dotted plot of peripheral blood samples in the presence of CD20-SN v605 single color conjugate in a buffer composition containing BSA and sodium azide alone as additives is shown in fig. 17C. In the absence of surfactant, granulocyte nonspecific staining was 1.20% and monocyte nonspecific staining was 1.63%.

A test dotted plot of peripheral blood samples in the presence of CD20-SN v605 single color conjugate in a buffer composition comprising BSA, sodium azide and NLS (0.16% w/v) as additives is shown in fig. 17D. Compared to the negative control (fig. 17C), the nonspecific staining of granulocytes was significantly reduced to 0.60%, and the nonspecific staining of monocytes was significantly reduced to 0.39%.

A test dotted plot of peripheral blood samples in the presence of CD20-SN v605 single color conjugate in a buffer composition comprising BSA, sodium azide and NLS (0.08% w/v) as additives is shown in fig. 17E. Compared to the negative control (fig. 17C), the nonspecific staining of granulocytes was reduced to 0.76% and the nonspecific staining of monocytes was reduced to 0.93%.

When compared with fig. 17D and 17E (NLS), fig. 17B (Empigen) The dot plots of (c) show that the% populations in the gates "Mons non-specific binding" and "Grans non-specific binding" are very similar, indicating that NLS is equivalent to Empigen at 0.16% and 0.08% in terms of effectiveness in preventing non-specific binding on cells.

An effective concentration of NLS was found to be 0.16% to 0.08% w/v to reduce or eliminate non-specific staining on monocytes and granulocytes in CD20-SN v605, thus indicating that this concentration range of NLS anionic surfactant is effective to reduce non-specific binding in monochromatic fluorescent polymer dye conjugate compositions.

Claims

1. 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 polymer dye conjugate with at least one zwitterionic or anionic surfactant prior to, during or after contacting the polymer dye conjugate with the blood sample, the contacting resulting in reduced non-specific binding of the at least one polymer dye conjugate to cells in the biological sample.

2. The method of claim 1, wherein the reduced non-specific binding comprises reduced non-specific binding to leukocytes in the biological sample; optionally, wherein the biological sample is a blood sample.

3. The method of claim 2, wherein the white blood cells are selected from monocytes and granulocytes.

4. A method according to any one of claims 1 to 3, comprising contacting the surfactant with the biological sample prior to contacting the polymer dye conjugate with the biological sample.

5. A method according to any one of claims 1 to 3, comprising contacting the polymer dye conjugate with a surfactant prior to contacting the polymer dye conjugate and surfactant with the biological sample.

6. The method of claim 1, wherein the surfactant is a compound of 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 ^- wherein

R ^1’ Is saturated or unsaturated C _5-24 An alkyl group;

x is NH, NR ^4’ Wherein R is ^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 is ^3’ Independently C _1-4 An alkyl group;

k is 0 or 1;

f is an integer from 0 to 4;

h is 0 or 1; and is also provided with

Y is COO, SO ₃ 、OPO(OR ^5’ ) O OR P (O) (OR) ^5’ ) O, where R is ^5’ Is H or C _1-4 An alkyl group.

7. The method of any one of claims 1 to 6, wherein the surfactant is a zwitterionic surfactant compound of the formula:

R ^1′ [CO-X(CH ₂ ) _j ] _g -N ⁺ (R ^2’ )(R ^3’ )-(CH ₂ ) _f -[CH(OH)CH ₂ ] _h -Y ^- ，

Wherein:

R ^1’ is saturated or unsaturated C _5-24 An alkyl group;

x is NH or NR ^4’ Wherein R is ^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 is ^3’ Independently C _1-4 An alkyl group;

f is an integer from 1 to 4;

h is 0 or 1; and is also provided with

Y is COO, SO ₃ 、OPO(OR ^5’ ) O OR P (O) (OR) ^5’ ) O, where R is ^5’ Is H or C _1-4 Alkyl residues.

8. The method of claim 7, wherein the zwitterionic surfactant is a compound 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 ^- the method comprises the steps of carrying out a first treatment on the surface of the Or (b)

R ^1′ -CO-NH-(CH ₂ ) ₃ -N ⁺ (CH ₃ ) ₂ -CH ₂ CH(OH)CH ₂ SO ₃ ^-

9. The method according to any one of claim 1 to 8, wherein the surfactant is selected from the group consisting of almond, wild apricot, avocado, babassu, behenamide, canola, capryl/decylamide, carnitine, cetyl, cocoamidoethyl, cocoamidopropyl hydroxysulfobetaine, coco betaine, cocohydroxysulfobetaine, cocohydroxy sulfobetaine, coco/oleamidopropyl betaine, coco sulfobetaine, decyl betaine, dihydroxyethyl glycinate, dihydroxyethyl soybean glycinate, dihydroxyethyl stearyl glycinate, dihydroxyethyl tallow glycinate, dimethicone propyl PG-betaine erucamide propyl hydroxysulfobetaine, hydrogenated tallow betaine, isostearamide propyl betaine, lauramidopropyl betaine, lauryl hydroxysulfobetaine, laurylsulfobetaine, lactamidopropyl betaine, myristamidopropyl betaine, oleamidopropyl hydroxysulfobetaine, oleyl betaine, olive oleamidopropyl betaine, palm oleamidopropyl betaine, palmamidopropyl betaine, palmitoyl carnitine, palm kernel oleamidopropyl betaine, polytetrafluoroethylene acetoxypropyl betaine, ricinoleamidopropyl betaine, sesame amidopropyl betaine, soybean amidopropyl betaine, stearamidopropyl betaine, stearyl betaine, tallow amidopropyl hydroxysulfobetaine, tallow betaine, tallow dihydroxyethyl betaine, undecamidopropyl betaine, and wheat germ amidopropyl betaine.

10. The method of any one of claims 1 to 9, wherein the surfactant is lauryl betaine.

11. The method of any one of claims 1 to 6, wherein the surfactant is an anionic surfactant compound of the formula:

R ^1′ [CO-X(CH ₂ ) _j ] _g -(CH ₂ ) _f -[CH(OH)CH ₂ ] _h -Y ^- wherein

R ^1’ Is saturated or unsaturated C _5-24 An alkyl group;

x is NH, NR ^4’ Wherein R is ^4’ Is C _1-4 Alkyl, O or S;

j is an integer from 1 to 10;

g is 0 or 1;

f is an integer from 0 to 4;

h is 0 or 1; and is also provided with

Y is COO, SO ₃ 、OPO(OR ^5’ ) O OR P (O) (OR) ^5’ ) O, where R is ^5’ Is H or C _1-4 Alkyl, and wherein the anionic surfactant may be in an acidic form or in the sodium or potassium salt form thereof.

12. The method of claim 11, wherein the anionic surfactant is a compound according to the formula:

R ^1′ -CO-N(CH ₃ )-CH ₂ -SO ₃ -, and the anionic surfactant is an acidic form or a sodium or potassium salt thereof, wherein

R ^1’ Is saturated or unsaturated C _5-24 An alkyl group.

13. The method of claim 11 or 12, wherein the anionic surfactant is selected from the group consisting of N-lauroyl sarcosine, sodium palmitoyl sarcosine, sodium stearoyl sarcosine, sodium N-methyl-N- (1-oxotetradecyl) -glycine, sodium caproyl sarcosine, sodium octanoyl sarcosine, N-methyl-N- (1-oxo-9-octadecen-1-yl) -glycine, sodium salt, sodium oleoyl sarcosine, and sodium linoleoyl sarcosine.

14. The method of claim 13, wherein the anionic surfactant is N-lauroyl sarcosine or a salt thereof.

15. The method of claims 1-14, wherein the polymer dye conjugate comprises a binding partner conjugated to a polymer dye having the structure of formula III:

wherein,,

each optional M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene, and ethynylene, and is uniformly or randomly distributed along the polymer backbone;

each optional L is a linker moiety;

a. c and d define the mole% of each unit, each of which may be uniform or randomly repeating, and wherein each a is 10% to 100% mole%, each c is 0 to 90% mole%, and each d is 0 to 25% mole%;

each b is independently 0 or 1; and is also provided with

Each m is an integer from 1 to about 10,000.

16. The method of claim 15, wherein a is selected from the group consisting of DHP moieties, fluorene moieties, and DHP and fluorene moieties.

17. The method of any one of claims 1 to 16, wherein the at least one polymer dye conjugate comprises a binding partner conjugated to a polymer having a structure according to formula I:

wherein the method comprises the steps of

Each X is independently C or Si;

each Y is independently CR ¹ R ² Or SiR ¹ R ² ；

each Z is independently selected from C, O and N;

Each subscript n is independently an integer of from 0 to 20;

each M is a unit capable of varying the band gap of the polymer and is uniformly or randomly distributed along the polymer backbone;

L is a linker;

G ¹ and G ² Each independently selected from hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen substituted aryl, silyl, diazonium salt, triflate, acetoxy, azide, sulfonate, phosphate, borate substituted aryl, borate, boric acid, optionally substituted Dihydrophenanthrene (DHP), optionally substituted fluorene, aryl or heteroaryl, substituted with one or more side chains terminated with a functional group selected from the group consisting of: amines, carbamates, carboxylic acids, carboxylic esters, maleimides, activated esters, N-hydroxysuccinimides, hydrazines, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof;

a. c and d define the mole% of each unit within the structure, each of which may be uniform or randomly repeating, and wherein each a is 10% to 100% mole%, each c is 0 to 90% mole%, and each d is 0 to 25% mole%;

each b is independently 0 or 1;

m is an integer from 1 to about 10,000; and is also provided with

Each n is independently an integer from 1 to 20.

18. The method of claim 17, wherein

L is aryl or heteroaryl, uniformly or randomly distributed along the polymer backbone, and is substituted with one or more side chains terminated with a functional group selected from the group consisting of: amines, carbamates, carboxylic acids, carboxylic acid esters, maleimides, activated esters, N-hydroxysuccinimide groups, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof.

19. The method of any one of claims 15 to 18, wherein the binding partner is a molecule or a molecular complex capable of specifically binding to a target analyte.

20. The method of any one of claims 15 to 19, wherein the binding partner is a protein, an affinity ligand, an antibody or an antibody fragment.

21. The method of claim 20, wherein the binding partner is selected from the group consisting 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.

22. A composition comprising

A polymer dye conjugate;

An aqueous buffer; and

zwitterionic surfactants or anionic surfactants.

23. The composition of claim 22, further comprising a non-polymeric dye conjugate.

24. The composition of claim 22 or 23, wherein the concentration of the zwitterionic surfactant or anionic surfactant is below a Critical Micelle Concentration (CMC).

25. The composition of claim 24, wherein 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).

26. The composition according to any one of claims 22 to 25, wherein the aqueous buffer comprises one or more further additives selected from protein stabilizers, preservatives and further surfactants.

27. The composition of any one of claims 22-26, wherein the polymer dye conjugate exhibits reduced non-specific binding to cells in the sample after contacting the composition with a biological sample as compared to non-specific binding of the polymer dye conjugate to cells in the sample when the polymer dye conjugate is contacted with the sample in the absence of a zwitterionic surfactant or an anionic surfactant.

28. The composition of claim 27, wherein the sample is a blood sample and the cells are leukocytes selected from monocytes and granulocytes.

29. The composition of any one of claims 22 to 28, wherein the surfactant is a compound of the formula:

R ^1’ Is saturated or unsaturated C _5-24 An alkyl group;

x is NH, NR ^4’ Wherein R is ^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 is ^3’ Independently C _1-4 An alkyl group;

k is 0 or 1;

f is an integer from 0 to 4;

h is 0 or 1; and is also provided with

Y is COO, SO ₃ 、OPO(OR ^5’ ) O OR P (O) (OR) ^5’ ) O, where R is ^5’ Is H or C _1-4 Alkyl group, andwhen k=0, the surfactant may be in an acidic form, or a sodium or potassium salt thereof.

30. The composition of any one of claims 22 to 29, wherein the polymer dye conjugate comprises a binding partner conjugated to a polymer dye having the structure of formula III:

wherein,,

Each optional L is a linker moiety;

each b is independently 0 or 1;

and each m is an integer from 1 to about 10,000.

31. The composition of any one of claims 22 to 30, wherein the polymer dye conjugate comprises a binding partner conjugated to a polymer dye having a structure according to formula I:

wherein the method comprises the steps of

Each X is independently C or Si;

each Y is independently CR ¹ R ² Or SiR ¹ R ² ；

each Z is independently selected from C, O and N;

Each subscript n is independently an integer of from 0 to 20;

l is a linker;

G ¹ and G ² Each independently selected from hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen substituted aryl, silyl, diazonium salt, triflateAn acetoxy group, an azide, a sulfonate, a phosphate, a borate substituted aryl, a borate, a boric acid, an optionally substituted Dihydrophenanthrene (DHP), an optionally substituted fluorene, an aryl or a heteroaryl group, substituted with one or more side chains terminated with a functional group selected from the group consisting of: amines, carbamates, carboxylic acids, carboxylic esters, maleimides, activated esters, N-hydroxysuccinimides, hydrazines, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof;

each b is independently 0 or 1;

m is an integer from 1 to about 10,000; and is also provided with

Each n is independently an integer from 1 to 20.