WO2024241780A1 - Method for detecting quantity of first structure and quantity of second structure - Google Patents

Abstract

[Problem] To provide a method for detecting the quantity of a first structure and the quantity of a second structure, the method comprising a step for detecting the quantity of a bonded first label and the quantity of a bonded second label together. [Solution] Provided is a method for detecting the quantity of a first structure and the quantity of a second structure, the method comprising: a step for bonding a first label to a first structure; a step for releasing a second structure from the first structure; a step for boding a second label to the second structure; and a step for detecting the quantity of the bonded first label and the quantity of the bonded second label together, wherein the second structure is included in the first structure, and the first label and the second label can be detected together, but are different.

Description

Method for detecting an amount of a first structure and an amount of a second structure

The present invention relates to a method for detecting the amount of a first structure and the amount of a second structure, a method for evaluating the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid, and a kit for use in these methods.

It is known that viral vectors used in gene therapy and other applications produce both active (containing DNA, etc.) particles (complete particles) and inactive (not containing DNA, etc.) particles (empty particles) during production. In addition, since it is difficult to completely separate these two types of particles through purification, viral vector products usually contain both complete and empty particles. The concentration and ratio of complete particles and empty particles are related to the efficacy and safety of viral vectors, and therefore need to be evaluated not only at the time of shipment but also during the development of manufacturing methods (Non-patent literature 1: Werle AK, Powers TW, Zobel JF, et al. Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors. Mol Ther Methods Clin Dev. 2021;23:254-262. Published 2021 Sep 1. doi:10.1016/j.omtm.2021.08.009).

Until now, the total particle concentration (complete and empty particles) was determined by the enzyme-linked immunosorbent assay (ELISA), and the nucleic acid concentration was quantified by the polymerase chain reaction (PCR) to estimate the complete particle concentration (Non-Patent Document 2: https://darkhorseconsultinggroup.com/wp-content/uploads/2022/05/DHC_Proposed-DRAFT-Guidance-for-FDA-Consideration.pdf). This method has concerns such as errors in the complete particle/empty particle ratio due to the combination of two different mechanisms, and the need to quantify complete particles and empty particles using samples that are not strictly the same. In addition, performing two measurements requires time and effort. In addition to the combination of ELISA and PCR, other methods such as ultracentrifugation and chromatography have been proposed to separate and quantify complete particles and empty particles, but these have limitations such as the need for prior purification when used in analysis for manufacturing method development. In addition, ultracentrifugation requires expensive equipment and skilled techniques.

Werle AK, Powers TW, Zobel JF, et al. Comparison of analytical techniques to quantitate the capsid content of adeno-ass associated viral vectors. Mol Ther Methods Clin Dev. 2021;23:254-262. Published 2021 Sep 1. doi:10.1016/j.omtm.2021.08.009 https://darkhorseconsultinggroup.com/wp-content/uploads/2022/05/DHC_Proposed-DRAFT-Guidance-for-FDA-Consideration.pdf

However, conventional methods for detecting the amount of a first label bound to a first structure and the amount of a second label bound to a second structure, or methods for evaluating the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid, require a step of separately detecting the amount of the first structure or viral capsid and the amount of the second structure or nucleic acid, respectively. Therefore, there is a need for a method for simultaneously detecting the amount of the first label bound to a first structure or viral capsid and the amount of the second label bound to a second structure or nucleic acid.

The method of the present invention for detecting the amount of a first structure and the amount of a second structure includes the steps of binding a first label to the first structure, releasing the second structure from the first structure, binding a second label to the second structure, and jointly detecting the amount of the bound first label and the amount of the bound second label, where the second structure is contained within the first structure, and the first label and the second label are different but can be detected together.

The method for assessing the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid according to the present invention includes the steps of immobilizing the viral capsid on a support, indirectly binding a first label to the viral capsid via an antibody that binds to the viral capsid, washing away the unbound first label, releasing nucleic acid from the viral capsid, binding a second label to the released nucleic acid, detecting the amount of the bound first label and the amount of the bound second label together, and calculating or determining the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid based on the amount of the bound first label and the amount of the bound second label, wherein the nucleic acid is encapsulated in the viral capsid, and the first label and the second label can be detected together but are different.

The kit according to the present invention includes a first label for a first structure, a second label for a second structure, and instructions for detecting the amount of bound first label and the amount of bound second label together, where the second structure is contained within the first structure, and the first label and the second label are different but can be detected together.

That is, the present invention provides the following:
[1] A method for detecting an amount of a first structure and an amount of a second structure, the method comprising:
Binding a first label to the first structure;
Releasing the second structure from the first structure;
binding a second label to the second structure and jointly detecting the amount of bound first label and the amount of bound second label;
Including,
a second structure is contained within the first structure, and the first label and the second label are detectable together but are different;
method.
[2] The method according to [1], further comprising the step of washing away the first label that has not bound to the first structure.
[3] The method according to [2], wherein a step of washing away the first label that has not bound to the first structure is performed after the step of binding the first label to the first structure and before the step of releasing the second structure from the first structure and the step of binding the second label to the second structure.
[4] The method according to [1], further comprising a step of fixing the first structure to a support.
[5] The method according to [1], wherein the first structure is composed of a protein, a lipid or a polymer, or a combination thereof.
[6] The method according to [1], wherein the first structure is selected from the group consisting of a virus particle, a virus capsid, a liposome, a lipid nanoparticle (LNP), and a polymer particle.
[7] The method according to [6], wherein the virus is selected from the group consisting of adeno-associated virus (AAV), retrovirus, lentivirus, and adenovirus.
[8] The method according to [1], wherein the second structure is composed of a nucleic acid, a protein or a compound, or a combination thereof.
[9] The method according to [8], wherein the second structure is selected from the group consisting of DNA, single-stranded DNA (ssDNA), self-complementary DNA (scDNA), RNA, miRNA, lncRNA, peptides, antibodies, detection compounds and drugs.
[10] The method according to [1], wherein the first label and the second label are fluorescent dyes or radioactive labels.
[11] The method according to [1], wherein the step of binding the first label to the first structure comprises indirect binding via a molecule that specifically binds to one or more first structures, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide, or comprises indirect binding via the molecule that specifically binds to the first structure and a secondary antibody that specifically binds to the molecule.
[12] The method of [1], wherein the step of jointly detecting the amount of bound first label and the amount of bound second label comprises detecting them together using one device or detector.
[13] The method according to [1], wherein the amount of the first structure and the amount of the second structure are calculated or determined based on the amount of the bound first label and the amount of the bound second label.
[14] The method of [1], further comprising calculating or determining a ratio of first structures that include a second structure to first structures that do not include a second structure.
[15] A method for evaluating a ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid, the method comprising:
Immobilizing the viral capsid on a support;
Indirectly binding a first label to the viral capsid via an antibody that binds to the viral capsid;
washing away unbound first label;
Releasing nucleic acid from the viral capsid;
binding a second label to the released nucleic acid;
detecting the amount of bound first label and the amount of bound second label together, and calculating or determining the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid based on the amount of bound first label and the amount of bound second label;
Including,
the nucleic acid is encapsulated in a viral capsid, and the first label and the second label are detectable together but are different,
method.
[16] a first marker for a first structure;
a second label on the second structure and instructions for jointly detecting the amount of bound first label and the amount of bound second label;
A detection kit comprising:
a second structure is contained within the first structure, and the first label and the second label are detectable together but are different;
Detection kit.

The method of the present invention includes a step of detecting the amount of the first label bound to the first structure and the amount of the second label bound to the second structure together. As a result, compared to a case where the method of the present invention includes a step of separately detecting the amount of the first label bound to the first structure and the amount of the second label bound to the second structure, the method of the present invention can, for example, reduce the time or effort required to detect the amount of the first structure and the amount of the second structure, reduce the cost of detecting the amount of the first structure and the amount of the second structure, reduce the detection error of the amount of the first structure and the amount of the second structure, eliminate the need to purify the first structure or the second structure from a sample containing impurities, or make it possible to detect the amount of the first structure and the amount of the second structure at low concentrations.

1 is a simplified flow chart of one embodiment of the method of the present invention. 1 is a simplified flow chart of one embodiment of the method of the present invention. Test results from Example 1 using AAV8. Test results from Example 2 using AAV2. Test results from Example 3 using a mixture of AAV2 and AAV8. Test results from Example 4 using Quantifluor. Test results from Example 5 using AAV2 containing ssDNA and AAV2 containing scDNA. Test results according to Example 6 using conventional wavelengths of 495 nm and 535 nm and wavelengths of 500 nm and 535 nm by the inventors. Test results from Example 7 using clear and black plates. Applicability of dFLISA to the analysis of unpurified samples. (A) Quantification of capsid titer. (B) Quantification of genome titer. (C) Percentage of intact particles. Different dilution factors for spike recovery were evaluated by comparing the experimental values obtained by dFLISA with the predicted values obtained by AUC analysis. The predicted values obtained from AUC were compared with the dFLISA values for three parameters: capsid titer, genome titer, and FP/EP ratio. Results are the average from duplicate wells. H is high spike, M is medium spike, and L is low spike. All data are presented as mean and standard deviation (n = 2). Determination of the ability of dFLISA to quantify unpurified samples. AAV8 content in unpurified samples was analyzed by three methods: dFLISA, ELISA, and dPCR. dFLISA was used to quantify capsid and genome titers and to calculate the FP/EP ratio of AAV8 content in unpurified samples. ELISA was used to quantify capsid content with SD calculated from independent measurements (n = 2). dPCR was used to determine genome content in unpurified samples with SD obtained from three independent measurements (n = 3). Complete particle ratio was calculated as dPCR/ELISA. All data presented in these experiments are the average of replicates for each assay. Comparison of fluorescence intensity of AAV with different genome lengths. The fluorescence intensity of ssDNA with different lengths obtained from AGE was divided by the MP result (relative area (concentration)) and plotted against the length of the ssDNA ladder standard.

In one embodiment, the present invention provides a method for detecting the amount (or concentration) of a first structure and the amount (or concentration) of a second structure, the method comprising the steps of binding a first label to the first structure, binding a second label to the second structure, and jointly detecting the amount of bound first label and the amount of bound second label, wherein the first label and the second label can be detected together but are different.

In one embodiment, the present invention provides a method for detecting the amount (or concentration) of a first structure and the amount (or concentration) of a second structure, the method comprising the steps of binding a first label to the first structure, releasing the second structure from the first structure, binding a second label to the second structure, and jointly detecting the amount (or concentration) of the bound first label and the amount (or concentration) of the bound second label, wherein the second structure is contained within the first structure, and the first label and the second label are different but can be detected together.

In one embodiment, the present invention provides a method for evaluating the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid, the method comprising the steps of immobilizing the viral capsid on a support, indirectly binding a first label to the viral capsid via an antibody that binds to the viral capsid, washing away the unbound first label, releasing nucleic acid from the viral capsid, binding a second label to the released nucleic acid, detecting the amount (or concentration) of the bound first label and the amount (or concentration) of the bound second label together, and calculating or determining the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid based on the amount (or concentration) of the bound first label and the amount (or concentration) of the bound second label, wherein the nucleic acid is encapsulated in the viral capsid, and the first label and the second label can be detected together but are different.

In one embodiment, the present invention provides a kit comprising a first label on a first structure, a second label on a second structure, and instructions for detecting the amount (or concentration) of the bound first label and the amount (or concentration) of the bound second label together, where the second structure is contained within the first structure, and the first label and the second label are different but can be detected together.

In this specification, the "first structure" may be composed of any material or substance, for example, a protein, lipid, or polymer, or a combination thereof, for example, a virus particle, a virus capsid, a liposome, a lipid nanoparticle (LNP), and a polymer particle. The "first structure" may be used as a tool for treating or diagnosing a disease or for in vivo gene transfer. The "first structure" may include a "second structure". The "first structure" may not include a "second structure". The "first structure" may be a "first structure" that includes a "second structure" and/or a "first structure" that does not include a "second structure". The "first structure" may be bound to a "second structure". The "first structure" may not be bound to a "second structure". The "first structure" may be a "first structure" that is bound to a "second structure" and/or a "first structure" that is not bound to a "second structure."

Viruses include any virus, such as adeno-associated viruses (AAV), retroviruses, lentiviruses and adenoviruses, and those viruses with modified viral capsid proteins. AAV includes natural adeno-associated viruses or recombinant adeno-associated viruses. AAV includes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11.

Adeno-associated virus (AAV) belongs to the Parvoviridae family. The AAV genome consists of a linear single-stranded DNA molecule of approximately 4.7 kilobases (kb) and consists of two major open reading frames encoding the nonstructural Rep (replication) protein and the structural Cap (capsid) protein. Recombinant vectors derived from AAV are used as tools for disease treatment or diagnosis, etc. Adeno-associated virus (AAV) derived viral vectors are used as tools for in vivo gene transfer due to their superior in vivo efficiency, broad tissue tropism and excellent safety profile compared to other vectors. Active (including DNA, etc.) adeno-associated virus (AAV) particles (complete particles) may be used as tools for disease treatment or diagnosis or in vivo gene transfer.

In this specification, the "second structure" may be composed of any material or ingredient, for example, a nucleic acid, a protein, or a compound, or a combination thereof, for example, DNA, single-stranded DNA (ssDNA), self-complementary DNA (scDNA), RNA, miRNA, lncRNA, peptide, antibody, detection compound, and drug. The "second structure" includes, for example, those that can be applied to the treatment, prevention, or diagnosis of a disease. The disease may be cancer, an autoimmune disease, an infectious disease, etc. The "second structure" may be an introduced gene, a transcription control gene, a gene therapy agent, a nucleic acid encoding a small molecule, a nucleic acid encoding an antibody, a nucleic acid encoding a protein, a small interfering RNA, an antisense RNA, a ribozyme, a nucleic acid encoding a therapeutic protein, a nucleic acid for genome editing, a nucleic acid for gene recombination, and a microRNA (miRNA), etc. The "second structure" may be included in the "first structure". The "second structure" may be bound to the "first structure".

In this specification, the term "encompassing" in the context of "a second structure is contained in a first structure," "a first structure containing a second structure," or "a first structure not containing a second structure" means a structure or state in which a first structure contains a second structure. For example, "a second structure is contained in a first structure" means that a second structure is present inside a first structure. The second structure may or may not be attached to the inner surface of the first structure. For example, "a first structure not containing a second structure" means that a second structure is not present inside the first structure.

As used herein, the term "label" includes any detectable signal source, such as fluorescent, visible, optical, colorimetric, dyes, enzymes, GCMS tags, avidin, biotin, and radioactive substances (radioactive labels and radiopaque, etc.). The term "label" includes various molecules such as dyes (fluorescent dyes) that emit fluorescent signals, radioactive labels, molecules that emit MRI signals, and molecules that emit ultrasound signals, as well as various microparticles such as iron oxide microparticles, gold microparticles, gold nanorods, and microparticles of metals and metal oxides such as platinum and silver. The "label" can be detected by an apparatus or detector based on the properties, activity, strength, or amount of the label. Those skilled in the art can appropriately select the label, apparatus, or detector based on the properties, activity, strength, or amount of the label. The "label" may be only a detectable signal source, or may be a protein, antibody, peptide, probe, etc. that has a detectable signal source.

The fluorescent dye may be any dye that emits a fluorescent signal, such as commercially available fluorescent dyes, known fluorescent dyes, Alexa Fluor (registered trademark) 647, SYBR (registered trademark) Gold, SYBR (registered trademark) green, QuantiFluor (registered trademark) ssDNA Dye Systems, fluorescein, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, SYBR Gold, SYBR Green I, SYBR Green II, SYBR Safe, or QuantiFluor ssDNA Dye Systems (QuantiFluor dsDNA, RNA or ssDNA System). The fluorescent dye can be detected by a light sensor, a light detector, a microscope, a device or a detector, etc., based on the wavelength, characteristic, activity, intensity or amount of the fluorescent signal or fluorescent dye. Those skilled in the art can appropriately select an optical sensor, optical detector, microscope, device, detector, etc. based on the wavelength, characteristics, activity, intensity, or amount of the fluorescent signal or fluorescent dye.

Radioactive labels include any label that emits radioactive substances, for example, commercially available radioactive labels, known radioactive labels, 18F, 32P, 35S, 129I, 131I, 64Cu, or 67Cu. Radioactive labels can be detected by a radiation or radioactivity sensor, radiation or radioactivity detector, device, or detector, etc., based on the characteristics, activity, strength, or amount of the radioactive label. Those skilled in the art can appropriately select a radiation or radioactivity sensor, radiation or radioactivity detector, device, or detector, based on the characteristics, activity, strength, or amount of the radioactive label.

As used herein, a "first label for a first structure" or a "first label" is directly or indirectly bound to a first structure. A "first label for a first structure" or a "first label" may be indirectly bound to a first structure via a molecule that specifically binds to one or more first structures, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide. A "first label for a first structure" or a "first label" may be indirectly bound to a first structure via an antibody that specifically binds to the first structure and a secondary antibody that specifically binds to the antibody. A "first label for a first structure" or a "first label" may be a label that is not destroyed or malfunctioned, or is not easily destroyed or malfunctioned, in a method that includes a step of releasing a second structure from a first structure. The "first label for the first structure" or the "first label" may be a label that is not destroyed or disabled, or is not easily destroyed or disabled, in the step of washing away the first label that is not bound to the first structure when the method includes the step of washing away the first label that is not bound to the first structure. The first structure may have one or more additional labels attached to it in addition to the "first label for the first structure" or the "first label". The "first label for the first structure" or the "first label" may be a fluorescent dye or a radioactive label. The "first label for the first structure" or the "first label" may be, for example, a commercially available fluorescent dye, a known fluorescent dye, Alexa Fluor (registered trademark) 647, fluorescein, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 5, or a similar fluorescent dye. 46, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, or TMB (3,3',5,5'-tetramethylbenzidine). Those skilled in the art can appropriately select the "first label for the first structure" or the "first label" based on the characteristics of the first structure, the first label, the device, the detector, or the detection result.

As used herein, the "second label for the second structure" or "second label" is directly or indirectly bound to the second structure. The "second label for the second structure" or "second label" may be indirectly bound to the second structure via one or more molecules that specifically bind to the second structure, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide. The "second label for the second structure" or "second label" may be indirectly bound to the second structure via an antibody that specifically binds to the second structure and a secondary antibody that specifically binds to the antibody. The "second label for the second structure" or "second label" may be a label that generates a different detectable signal when bound to the second structure compared to when not bound to the second structure. The "second label for the second structure" or "second label" may be a label that generates a detectable signal when bound to the second structure, but does not generate a detectable signal when not bound to the second structure. A "second label for a second structure" or a "second label" may be a label that generates a greater or lesser or different detectable signal when bound to the second structure than when not bound to the second structure. The second structure may have one or more additional labels bound to it in addition to the "second label for a second structure" or the "second label". A "second label for a second structure" or a "second label" may be a fluorescent dye or a radioactive label. The "second label for the second structure" or the "second label" may be, for example, a commercially available fluorescent dye, a known fluorescent dye, SYBR (registered trademark) Gold, SYBR (registered trademark) green, QuantiFluor (registered trademark) ssDNA Dye Systems, fluorescein, SYBR Gold, SYBR Green I, SYBR Green II, SYBR Safe, QuantiFluor ssDNA Dye Systems (QuantiFluor dsDNA, RNA, etc.) or ssDNA System), SYPRO orange, Thioflavin T, SYPRO orange, SYPRO Tangerine, SYPRO Red, SYPRO Ruby, SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, SYTOX AADvanced, or Hoechst, for example Hoechst 33343 and 33258. Those skilled in the art can appropriately select a "second label for the second structure" or a "second label" based on the characteristics of the second structure, the second label, the device, the detector, or the detection result.

In this specification, the "first label for the first structure" or the "first label" and the "second label for the second structure" or the "second label" are different labels that can be detected together. For example, the "first label" and the "second label" are labels that can be detected together using one or more devices or detectors, and a detection result of each label of the first structure and the second structure can be obtained. For example, the "first label" and the "second label" are fluorescent dyes that emit different fluorescent signals and can be detected together using one or more microscopes, devices or detectors, and a detection result of the fluorescent signal of each fluorescent dye of the first label and the second label can be obtained based on, for example, each wavelength. For example, the "first label" and the "second label" can be detected together using one device or detector. A person skilled in the art can appropriately select the "first label" and the "second label" based on the characteristics of the first structure, the second structure, the first label, the second label, the device, the detector, or the detection result.

As used herein, in the context of "detecting the amount of bound first label and the amount of bound second label together," "detecting together" means detecting two or more substances to be detected in one space or location that contains them (e.g., one sample, one container, or one well in a plate). For example, "detecting the amount of bound first label and the amount of bound second label together" may mean detecting the amounts of each of the first label and the second label in one space or location that contains the bound first label and the bound second label (e.g., one sample, one container, or one well in a plate). For example, "detecting the amount of bound first label and the amount of bound second label together" may mean detecting the amount of each of the first label and the second label simultaneously, or sequentially or sequentially (e.g., detecting the amount of each of the first label and the second label simultaneously, detecting the amount of the first label first and then the amount of the second label, or detecting the amount of the second label first and then the amount of the first label).

As used herein, a "kit" includes a first label for a first structure, a second label for a second structure, and instructions for detecting the amount of bound first label and the amount of bound second label together. The "kit" may be a kit for use in any method of detecting the amount of a first structure and the amount of a second structure described herein. The "kit" may further include any peptide, protein, antibody, nucleic acid, additional label, enzyme, substrate, reagent, buffer, salt, plate, or container, etc.

As used herein, "about" means approximately, or around. When used in conjunction with a numerical range, "about" may extend that range above and below the boundaries of the stated numerical values. "About" may mean a numerical range of ±10% of the stated numerical value.

In one embodiment, the method of the invention includes a step of immobilizing or binding the first structure and/or the second structure to a support before or after the step of binding the first label and/or the step of binding the second label. The first structure and/or the second structure may be indirectly bound to the support via one or more substances, such as a molecule that specifically binds to the first structure and/or the second structure, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide. The step of indirectly immobilizing the first structure and/or the second structure on the support may include (i) attaching to the support a substance capable of binding to the support and capable of binding to the first structure and/or the second structure, such as a molecule that specifically binds to the first structure and/or the second structure, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide, (ii) washing the support by using a washing buffer, (iii) coating the support by using a blocking buffer, (iv) washing the support by using a washing buffer, (v) binding the first structure and/or the second structure to the substance, such as a molecule, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide, and/or (vi) washing the support by using a washing buffer. For example, the step of immobilizing the first structure and/or the second structure on the support may hold or maintain the first structure and/or the second structure on the support. For example, the process of immobilizing the first structure and/or the second structure on the support allows unwanted substances, including buffer and substances that are not bound to the support, such as molecules, e.g., antibodies, antibody fragments, lectins, aptamers, ligands, peptides, or cyclic peptides, unbound first structure and/or unbound second structure, to be washed from the support.

The support includes any plate or vessel, e.g., a petri dish, microplate, or vessel for culture, measurement, or ELISA. The support may be of any color, e.g., transparent, translucent, white, or black. The support may be white for chemiluminescence detection or black for fluorescence or chemiluminescence detection. The support may be a black microplate for fluorescent dye detection.

In one embodiment, the method of the present invention includes a step of binding a first label to a first structure and a step of binding a second label to a second structure. The steps of binding the first label to the first structure and the second label to the second structure may be performed in any order or simultaneously. The steps of binding the first label to the first structure and the second label to the second structure may be performed by first binding the first label to the first structure and then binding the second label to the second structure. The steps of binding the first label to the first structure and the second label to the second structure may be performed by first binding the second label to the second structure and then binding the first label to the first structure.

In one embodiment, the step of binding the first label to the first structure includes directly or indirectly binding the first label to the first structure. The step of binding the first label to the first structure may include indirectly binding the first label to the first structure via one or more substances, such as a molecule that specifically binds to the first structure, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide. The step of binding the first label to the first structure may include indirectly binding the first label to the first structure via an antibody that specifically binds to the first structure. The step of binding the first label to the first structure may or may not include washing the support after directly or indirectly binding the first label to the first structure. The step of binding the first label to the first structure may include (i) attaching an antibody capable of specifically binding to the first structure to the first structure, (ii) washing the support by using a wash buffer, (iii) binding the first label to the antibody capable of specifically binding to said first structure, and/or (iv) washing the support by using a wash buffer. The step of binding the first label to the first structure may include indirectly binding the first label to the first structure via an antibody that specifically binds to the first structure and a secondary antibody that specifically binds to said antibody. The step of binding the first label to the first structure may include (i) attaching an antibody capable of specifically binding to the first structure (e.g., an antibody that specifically binds to a virus capsid derived from a mouse) to the first structure, (ii) washing the support by using a washing buffer, (iii) binding a secondary antibody that specifically binds to the antibody having the first label (e.g., an anti-mouse IgG antibody that specifically binds to the antibody derived from a mouse having Alexa Fluor (registered trademark) 647) to the antibody, and/or (iv) washing the support by using a washing buffer.

In one embodiment, the step of binding the second label to the second structure includes directly or indirectly binding the second label to the second structure. The step of binding the second label to the second structure may include indirectly binding the second label to the second structure via one or more substances, such as a molecule that specifically binds to the second structure, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide. The step of binding the second label to the second structure may include indirectly binding the second label to the second structure via a peptide that specifically binds to the second structure. The step of binding the second label to the second structure may or may not include washing the support after directly or indirectly binding the second label to the second structure. The step of binding the second label to the second structure may include binding a peptide (e.g., SYBR® Gold) that specifically binds to the second structure having the second label to the second structure, and/or (ii) not washing the unbound peptide from the sample. The step of binding the second label to the second structure may include (i) binding a peptide (e.g., SYBR® Gold) that specifically binds to the second structure having the second label to the second structure, and/or (ii) washing the unbound peptide from the sample by using a wash buffer.

In one aspect, the method of the present invention includes a step of releasing the second structure from the first structure when the second structure is contained in the first structure. The step of releasing the second structure from the first structure is performed, for example, by using a temperature change, heating, cooling, stirring, pipetting, a chemical, a pH change, an acid or an alkali. A person skilled in the art can appropriately select a step of releasing the second structure from the first structure based on the properties, activity, strength or amount of the "first structure" and the "second structure". For example, the heating may be performed at about 75°C to about 95°C, about 80°C to about 90°C, or about 85°C. For example, the heating may be performed for about 5 minutes to about 25 minutes, about 10 minutes to about 20 minutes, or about 15 minutes. For example, the heating may be performed at about 85°C for about 15 minutes. The step of releasing the second structure from the first structure may be performed before the step of binding the first label to the first structure. The step of releasing the second structure from the first structure may be after the step of binding the first label to the first structure. The step of releasing the second structure from the first structure may be before the step of binding the second label to the second structure. The step of releasing the second structure from the first structure may be after the step of binding the first label to the first structure and before the step of binding the second label to the second structure. For example, the step of releasing the second structure from the first structure makes it easier for the second label to bind to the second structure. For example, the step of releasing the second structure from the first structure allows the second label to bind to the second structure that was included in the first structure. For example, the step of releasing the second structure from the first structure allows the second label to bind to the second structure more accurately or with greater precision.

In one embodiment, the method of the present invention includes a step of detecting the amount of the bound first label and the amount of the bound second label together. The step of detecting the amount of the bound first label and the amount of the bound second label together includes detecting them together, for example, using one or more devices or detectors. The step of detecting the amount of the bound first label and the amount of the bound second label together includes detecting them together, for example, using one device or detector. For example, the step of detecting the amount of the bound first label and the amount of the bound second label together includes obtaining a detection result of each of the labels, the "first label" and the "second label". For example, the step of detecting the amount of the bound first label and the amount of the bound second label together includes obtaining a detection result of each of the fluorescent signals based on excitation and emission, when the "first label" and the "second label" are fluorescent dyes that emit different fluorescent signals. When the "first label" is Alexa Fluor® 647, the detection result of the fluorescent signal may be obtained based on a wavelength of 652 nm (excitation) and/or 680 nm (emission). When the "second label" is SYBR® Gold, the detection result of the fluorescent signal may be obtained based on a wavelength of 495 nm (excitation) and/or 535 nm (emission), or based on a wavelength of 500 nm (excitation) and/or 535 nm (emission). When the "first label" is Alexa Fluor® 647 and the "second label" is SYBR® Gold, for example, one optical sensor, photodetector, microscope, device or detector may be used to obtain detection results of fluorescent signals based on wavelengths of 652 nm (excitation) and/or 680 nm (emission) and detection results of fluorescent signals based on wavelengths of 495 nm (excitation) and/or 535 nm (emission) and/or 500 nm (excitation) and/or 535 nm (emission). The process of detecting the amount of bound first label and the amount of bound second label together, compared to the process of detecting the amount of bound first label and the amount of bound second label separately (not together), can, for example, reduce the time or effort required to detect the amount of the first structure and the amount of the second structure, reduce the cost of detecting the amount of the first structure and the amount of the second structure, reduce the detection error of the amount of the first structure and the amount of the second structure, eliminate the need to purify the first structure or the second structure from a sample containing contaminants, or make it possible to detect the amount of the first structure and the amount of the second structure at low concentrations.

In one embodiment, the method of the present invention includes a step of calculating or determining the amount (or concentration) of the first structure and the amount (or concentration) of the second structure based on the amount (or concentration) of the bound first label and the amount (or concentration) of the bound second label. A person skilled in the art can appropriately calculate or determine the amount (or concentration) of the first structure and the amount (or concentration) of the second structure based on the characteristics of the first structure, the second structure, the label, the device, the detector, or the detection result. For example, the step of calculating or determining the amount (or concentration) of the first structure and the amount (or concentration) of the second structure may include creating a calibration curve of the amount (or concentration) of the first label and the amount (or concentration) of the first structure and/or a calibration curve of the amount (or concentration) of the second label and the amount (or concentration) of the second structure. For example, a calibration standard (e.g., a commercially available standard, standard solution, and standard sample) may be used to create a calibration curve. Those skilled in the art can appropriately determine the standard for the calibration curve to be used based on the characteristics of the first structure, the second structure, the label, the device, the detector, or the detection result. For example, the standard for the calibration curve may be set for each of the first structure and/or the second structure to be calculated or determined. For example, even if the length of the first structure and/or the second structure is different from that of the standard, the amount (or concentration) of one or more first structures and/or the amount (or concentration) of one or more second structures can be calculated or determined by performing a correction by calculation. For example, even if the length of the second structure is different from that of the standard for the calibration curve, the amount (or concentration) of two or more second structures can be calculated or determined by using one type of standard for the calibration curve and performing a correction by calculation (for example, based on the correlation between DNA length and fluorescence intensity).

In one embodiment, when a second structure is contained in a first structure, the amount (or concentration) of the first structure containing the second structure (e.g., a viral capsid containing a nucleic acid) and/or the amount (or concentration) of the first structure not containing the second structure (e.g., a viral capsid not containing a nucleic acid) can be determined based on the calculated or determined amount (or concentration) of the first structure and the amount (or concentration) of the second structure. For example, the amount (or concentration) of the first structure containing the second structure (e.g., a viral capsid containing a nucleic acid) can be determined based on the calculated or determined amount (or concentration) of the second structure. For example, the amount (or concentration) of the first structure not containing the second structure (e.g., a viral capsid not containing a nucleic acid) can be determined based on the calculated or determined amount (or concentration) of the second structure from the calculated or determined amount (or concentration) of the first structure. Based on the calculated or determined amount (or concentration) of the first structure and the amount (or concentration) of the second structure, the ratio of the first structure containing the second structure (e.g., viral capsids containing nucleic acid) to the first structure not containing the second structure (e.g., viral capsids not containing nucleic acid) can be evaluated or determined. For example, based on dividing the calculated or determined amount (or concentration) of the second structure by the calculated or determined amount (or concentration) of the first structure, the percentage (proportion) of the first structure containing the second structure (e.g., viral capsids containing nucleic acid) can be determined. For example, based on such percentage (proportion), the percentage (proportion) of the first structure not containing the second structure (e.g., viral capsids not containing nucleic acid) can be determined. For example, based on the calculated or determined amount (or concentration) of the first structure and the amount (or concentration) of the second structure, the amount (or concentration) of the active viral vector (complete particle) and/or the amount (or concentration) of the inactive viral vector (empty particle) can be determined. For example, the ratio of full particles to empty particles can be evaluated or determined based on the calculated or determined amount (or concentration) of the first structure and the amount (or concentration) of the second structure.

In one embodiment, the method of the present invention may use ELISA (Enzyme-Linked Immunosorbent Assay), which is an immunological assay. The method of the present invention is an application of ELISA, which is an immunological assay, and is called Dual Fluorescence-Linked Immunosorbent Assay (dFLISA).

In one embodiment, the method of the present invention may include detecting the amount (or concentration) of one or more additional structures (e.g., a third structure and/or a fourth structure) in addition to detecting the amount (or concentration) of the first structure and the amount (or concentration) of the second structure. For example, a sample, a container, or a well in a plate may include one or more additional structures (e.g., a third structure and/or a fourth structure) in addition to the first structure and the second structure. For example, the step of detecting the amount (or concentration) of one or more additional structures (e.g., a third structure and/or a fourth structure) may be the same as the method or step of detecting the amount (or concentration) of the first structure and the amount (or concentration) of the second structure described herein.

Unless otherwise specified, the terms used in this specification are terms commonly used in the art.

The present invention will be explained in more detail in the following examples, but it should not be understood that the scope of the present invention is limited to these examples.

List of materials and equipment used

Example 1

Preparation of AAV VHH antibody The reagents were weighed out and mixed in a 15 mL tube (100-fold dilution). An example is shown below.

Coating of AAV VHH antibody onto plate 100 μL of diluted AAV VHH antibody was added to each well. An example is shown below.
A sticker was placed on the top surface of the plate and pressed into place with a spatula.
Incubated at 4°C for 16 hours.

Preparation of 1x PBS: Weigh out the reagents and mix them in a 500 mL glass bottle. An example is shown below.




The vial was shaken to mix well.

Preparation of 1% TWEEN20 in 1x PBS: Weigh out the reagents into a 15 mL tube and mix them. An example is shown below.




The tube was shaken and mixed well by vortexing.

Preparation of wash buffer (0.05% TWEEN20 in 1x PBS, pH 7.4) The reagents were weighed and mixed in a 500 mL glass bottle. An example is shown below.




The vial was shaken to mix well.

Preparation of blocking buffer and dilution buffer (1% BSA in 0.05% TWEEN20 of 1x PBS, pH= 7.0) The reagents were weighed and mixed in a 50 mL tube. An example is shown below.




Mix by vortexing.

Washing of unbound VHH antibodies The plate was removed from the refrigerator (4°C).
The VHH solution in each well of the plate was removed with a multichannel pipette.
After adding 200 μL of wash buffer to each well, the plate was turned over and the wash buffer was removed with a Kimtowel. This operation was repeated three times.

Blocking of the plate 200 μL of blocking buffer was added to each well.
A sticker was placed on the top surface of the plate and pressed into place with a spatula.
Incubated at 37°C for 1 hour.
After incubation, the blocking buffer in each well was removed with a multichannel pipette.
After adding 200 μL of wash buffer to each well, the plate was turned over and the wash buffer was removed with a Kimtowel. This operation was repeated three times.

Preparation of AAV standard and sample solutions <Preparation of calibration curve solutions>
The AAV sample for the standard curve was diluted in a 1.5 mL microtube. An example is shown below.

The diluted AAV standard curve sample was serially diluted as follows to prepare standard curve samples (SD1 to SD7).




<Preparation of sample solution for measurement>
The AAV sample for measurement was diluted in a 1.5 mL microtube. An example is shown below.

Addition of sample AAV 100 μL of sample AAV was added to each well. An example is shown below.




* B: Wash buffer SD: Standard curve sample
SP: Measurement sample

A sticker was placed on the top surface of the plate and pressed into place with a spatula.
Incubated at 37°C for 1 hour.
After incubation, the AAV solution in each well was removed with a multichannel pipette.
After adding 200 μL of wash buffer to each well, the plate was turned over and the wash buffer was removed with a Kimtowel. This operation was repeated three times.

Preparation of anti-AAV8 antibody (ADK8)
The reagents were weighed out and mixed in a 15 mL tube (50x). An example is shown below.




The tube was shaken to mix well.

Addition of anti-AAV8 antibody (ADK8) 100 μL of the prepared ADK8 was added to each well. A seal was attached to the top of the plate and pressed firmly with a spatula.
Incubated at 37°C for 1 hour.
After incubation, the AAV solution in each well was removed with a multichannel pipette.
After adding 200 μL of wash buffer to each well, the plate was turned over and the wash buffer was removed with a Kimtowel. This operation was repeated three times.

Preparation of Goat Anti-Mouse IgG H&L (Alexa Fluor 647) The reagents were weighed out and mixed in a 15 mL tube (500-fold dilution). An example is shown below.




The tube was gently shaken to mix well.
The 15 mL tube was covered with aluminum foil.

Addition of Goat Anti-Mouse IgG H&L (Alexa Fluor 647) 100 μL of Goat Anti-Mouse IgG H&L (Alexa Fluor 647) was added to each well.
A sticker was placed on the top surface of the plate and pressed into place with a spatula.
The mixture was incubated at 37°C for 1 hour in the dark at 300 rpm.
After incubation, the AAV solution in each well was removed with a multichannel pipette.
After adding 200 μL of wash buffer to each well, the plate was turned over and the wash buffer was removed with a Kimtowel. This operation was repeated three times.

Genome extraction 100 μL of 1x PBS was added to each well.
A sticker was placed on the top surface of the plate and pressed into place with a spatula.
Incubated at 85°C for 15 minutes.
After incubation, the plate was covered with aluminum foil and allowed to stand at room temperature for 5 minutes.

Preparation of SYBR Gold: The reagents were weighed out and mixed in a 15 mL tube (1000-fold dilution). An example is shown below.




The tube was gently shaken to mix well.
The 15 mL tube was covered with aluminum foil.

Addition of SYBR Gold 10 μL of SYBR Gold was added to each well.
The plate was gently tilted to mix.
Cover the plate with aluminum foil and let sit at room temperature for 5 minutes.

Fluorescence intensity was measured using a SpectraMax i3x.
Data Analysis: A 4-parameter logistic regression was performed on the results of the standard curve solution to create a standard curve. The Capsid concentration and nucleic acid concentration of each sample were determined from the standard curve.
Measurement conditions




The results are shown in Figure 2.

Example 2
The test was carried out in the same manner as in Example 1, except that AAV8 and anti-AAV8 antibody (ADK8) were replaced with AAV2 and anti-AAV2 antibody (A20) shown in the table below. The results are shown in Figure 3.

Example 3
AAV8 and anti-AAV8 antibody (ADK8) were tested as in Example 1, using a mixture of AAV2 and AAV8 as shown in the table below, and anti-AAV2 antibody (A20) and anti-AAV8 antibody (ADK8). The results are shown in Figure 4. A dose-response curve was obtained for the total number of AAV8 capsids. Detection of antibodies against individual AAV serotypes (AAV8 & AAV2) was confirmed by the method of the present invention (dFLISA).

Example 4
The test was carried out in the same manner as in Example 1, except that SYBR Gold was replaced with QuantiFluor, and proteins were detected using antibodies labeled with HRP (Horse Radish Peroxidase) and absorbance measurements were performed when TMB (3,3',5,5'-tetramethylbenzidine) was added. The results are shown in Figure 5.

Example 5
A test was performed in the same manner as in Example 2 using AAV2 containing ssDNA and AAV2 containing scDNA. The results are shown in Figure 6. The fluorescence intensity of AAV2 scDNA was about 1.4-1.6 times that of AAV2 ssDNA. Since scDNA is a long-chain DNA, it may be possible to perform quantification independent of DNA length by appropriately correcting for the DNA length.

Example 6
Measurement of DNA fluorescence intensity was performed in the same manner as in Example 2, using conventional wavelengths of 495 nm and 535 nm or wavelengths of 500 nm and 535 nm (optimized conditions) by the inventors. The results are shown in Figure 7. The signal background of the samples was equal or higher under the optimized conditions compared to the conventional wavelengths.

Example 7
Tests were performed in the same manner as in Example 2 using a transparent plate (Nunc MaxiSorpTM flat-bottom clear 96-well plate) and a black plate (The Nunc MaxiSorpTM flat-bottom black 96-well plate). The results are shown in FIG. 8. The fluorescence intensity of the sample showed a higher response in the black plate compared to the transparent plate. This result suggests that the black microplate is suitable for providing a signal suitable for the method of the present invention.

Example 8
To confirm whether the method of the present invention (dFLISA) can be used to analyze unpurified samples without purification, a spike recovery test was performed. The recovery rate was within ± 25% of the predicted value, meeting the criteria. This indicates that the dFLISA results are not easily affected by contaminants contained in unpurified samples. This indicates that dFLISA is an excellent method for evaluating unpurified AAV samples. The dFLISA method is a valuable and novel method that can be used to accurately quantify the titer of unpurified samples, and is unique in that it can directly quantify the full and empty capsid concentrations and FP/EP ratios of unpurified samples. The results are shown in Figure 9.

dFLISA can measure the capsid titer and genome titer of unpurified samples. The capsid titer measured by dFLISA was comparable to that measured by ELISA. However, the genome titer results by dFLISA were higher than those by dPCR. Considering the high recovery rate in the spike recovery experiment, the dFLISA results are considered reliable. Since previous studies have shown that dPCR can be affected by impurity interference, dFLISA may be a more accurate method to measure the concentration of AAV genome material in unpurified samples compared with the combination of ELISA and dPCR. The results are shown in Figure 10.

The results of dFLISA suggest that it is less affected by impurities present in unpurified samples and is a reliable analytical technique for AAV vector particle analysis.

Comparison of quantification of crude samples by dFLISA and other methods The dFLISA method was as described above. In these experiments, AAV8-Lot1 was diluted 60-fold in 1 x PBS (containing 0.05% Tween 20) to final concentrations of 2.01 x ¹⁰¹¹ cp/mL and 1.81 x ¹⁰¹¹ vg/mL. Serial dilutions were then performed at a 1:2 ratio to prepare calibration curve standards. For spike samples, AAV8-Lot2 was concentrated by ultracentrifugation to final concentrations of 5.36 x ¹⁰¹³ cp/mL and 4.75 x ¹⁰¹³ vg/mL, and then diluted as follows in 1 x PBS (containing 0.05% Tween 20): High concentration (Spike H): 1.07 x ¹⁰ cp/mL and 9.50 x ¹⁰ vg/mL, medium concentration (Spike M): 7.15 x ¹⁰ cp/mL and 6.33 x ¹⁰ vg/mL, low concentration (Spike L): 5.36 x ¹⁰ cp/mL and 4.75 x ¹⁰ vg/mL. Each spike sample solution was added to an unknown concentration unpurified sample (AAV8-Lot4) in a 1:1 volume ratio and mixed, and the prepared spike ratio sample solution was added to each well of the plate in 100 μL. N=2 analyses were performed. The recovery rate was evaluated using the following formula:
Mixed spike (HM) = (1/2) (crude + spike H) - (1/2) (crude + spike M) (Equation 3)
Mixed spike (HM) = (1/2) (crude + spike H) - (1/2) (crude + spike L) (Equation 4)
The target recovery rate (%) was set within ±25%.

Quantification of capsid and genome titers of unpurified samples. dFLISA was performed as described above. AAV8-Lot1 was diluted 60-fold in 1x PBS (containing 0.05% Tween 20) to a final concentration of 2.01 x ¹⁰¹¹ cp/mL and 1.81 x ¹⁰¹¹ vg/mL, and then serially diluted at a 1:2 ratio to serve as calibration curve standards. AAV8 unpurified samples of unknown concentration were then analyzed using dFLISA as detailed in the previous section (n=2).

Example 9
The fluorescence intensity in the quantification of genomes using fluorescent dyes varies depending on the length of the genome, which is also related to dFLISA. When the correlation with the fluorescence intensity normalized by concentration was compared using DNA of different genome lengths, a correlation was observed between genome length and fluorescence intensity. The results are shown in Figure 11. Furthermore, when the fluorescence intensity was compared between AAV vectors of different genome lengths (Table 22), the fluorescence intensity was high in samples with long genome lengths, and the ratio of genome length to fluorescence intensity was equivalent to that in Figure 11. From these results, the validity of dFLISA as a method for evaluating the genomes of AAV vectors with various nucleic acids was confirmed. In addition, even if the genome length is different from that of the standard AAV vector, the genome length can be corrected by calculation, and it was shown that it is not necessary to prepare a standard AAV for each AAV with a different genome length.

Fluorescence intensity of ssDNA from 1100 bp to 5100 bp was measured using ssDNA 7K ladder (PerkinElmer, Waltham, MA, USA). First, the ssDNA ladder marker solution was appropriately diluted with Milli-Q and then loaded onto a 1% agarose gel. Next, 10 μL of sample was added to 2 μL of EzApplyDNA (6x loading buffer), mixed thoroughly by pipetting, and 10 μL of the mixture was loaded onto the gel. Electrophoresis was performed at 70 V for 45 min. After electrophoresis, the gel was stained and washed according to the manufacturer's instructions. SYBR Gold Nucleic Acid Gel Staining solution was used to stain the gel. Quantitative analysis of brightness density in the stained gel was performed.

Then, mass photometry (MP) measurements were performed. Before MP measurements, the ssDNA ladder solution was diluted and analyzed at room temperature. Data analysis was performed using DiscoverMP.

Then, the fluorescence intensity of each ssDNA band obtained from the five agarose gel electrophoresis (AGE) was divided by the MP area (%) and plotted against the ssDNA length value. The expected intensity of the AAV vector carrying both scDNA (3681 bp) and ssDNA (2521 bp) was also estimated from the curve.

The fluorescence intensities of AAV containing ssDNA and scDNA were examined using dFLISA. AAV2-Lot1 containing ssDNA was diluted 100-fold in 1x PBS (containing 0.05% Tween 20) to final concentrations of 7.70 x ¹⁰¹⁰ cp/mL and 6.41 x ¹⁰¹⁰ vg/mL. A standard curve was prepared by serial dilution in 1:2 ratio steps. Similarly, AAV2-Lot2 containing scDNA was diluted 100-fold to final concentrations of 2.11 x ¹⁰¹¹ cp/mL and 1.93 x ¹⁰¹¹ vg/mL. A standard curve was prepared by serial dilution in 1:2 ratio steps. The fluorescence intensity ratios of ssDNA AAV8 and scDNA AAV2 were measured by dFLISA and compared with the fluorescence intensity ratios calculated from the curves.

Claims (16)

1. A method for detecting an amount of a first structure and an amount of a second structure, the method comprising:
Binding a first label to the first structure;
Releasing the second structure from the first structure;
binding a second label to the second structure and jointly detecting the amount of bound first label and the amount of bound second label;
Including,
a second structure is contained within the first structure, and the first label and the second label are detectable together but are different;
method. The method of claim 1, further comprising a step of washing away the first label that is not bound to the first structure. The method according to claim 2, wherein the step of washing away the first label that is not bound to the first structure is performed after the step of binding the first label to the first structure and before the step of releasing the second structure from the first structure and the step of binding the second label to the second structure. The method of claim 1, further comprising the step of fixing the first structure to a support. The method of claim 1, wherein the first structure is composed of a protein, a lipid, or a polymer, or a combination thereof. The method of claim 1, wherein the first structure is selected from the group consisting of a virus particle, a virus capsid, a liposome, a lipid nanoparticle (LNP), and a polymer particle. The method of claim 6, wherein the virus is selected from the group consisting of an adeno-associated virus (AAV), a retrovirus, a lentivirus, and an adenovirus. The method of claim 1, wherein the second structure is composed of a nucleic acid, a protein, or a chemical compound, or a combination thereof. The method of claim 8, wherein the second structure is selected from the group consisting of DNA, single-stranded DNA (ssDNA), self-complementary DNA (scDNA), RNA, miRNA, lncRNA, peptides, antibodies, detection compounds and drugs. The method of claim 1, wherein the first label and the second label are fluorescent dyes or radioactive labels. The method of claim 1, wherein the step of binding the first label to the first structure comprises indirectly binding via a molecule that specifically binds to one or more of the first structures, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide, or indirectly binding via the molecule that specifically binds to the first structure and a secondary antibody that specifically binds to the molecule. The method of claim 1, wherein the step of jointly detecting the amount of bound first label and the amount of bound second label comprises jointly detecting them using a single device or detector. The method of claim 1, wherein the amount of the first structure and the amount of the second structure are calculated or determined based on the amount of the bound first label and the amount of the bound second label. The method of claim 1, further comprising calculating or determining a ratio of first structures that contain second structures to first structures that do not contain second structures. 1. A method for assessing a ratio of viral capsids that contain nucleic acid to viral capsids that do not contain nucleic acid, the method comprising:
Immobilizing the viral capsid on a support;
Indirectly binding a first label to the viral capsid via an antibody that binds to the viral capsid;
washing away unbound first label;
Releasing nucleic acid from the viral capsid;
binding a second label to the released nucleic acid;
detecting the amount of bound first label and the amount of bound second label together, and calculating or determining the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid based on the amount of bound first label and the amount of bound second label;
Including,
the nucleic acid is encapsulated in a viral capsid, and the first label and the second label are detectable together but are different,
method. a first indicator for the first structure;
a second label on the second structure and instructions for jointly detecting the amount of bound first label and the amount of bound second label;
A detection kit comprising:
a second structure is contained within the first structure, and the first label and the second label are detectable together but are different;
Detection kit.