Method for determining DNA concentration in DNA virus

Abstract

The present invention relates to a method for determining the concentration of DNA in a DNA virus. The invention is characterized by three basic steps. The initial step is to capture a defined amount of viral capsid particles on a first solid phase. The second step is cleavage of the capsid to release the viral DNA from the first solid phase into a cleavage solution. After separating the lysis solution from the first solid phase, a third step is contacting the lysis solution with a second solid phase. The second solid phase captures total DNA derived from the captured capsid. The invention also relates to a method for measuring the percentage of intact viral capsid, comprising first determining the ssDNA concentration in the virus and then converting the ssDNA concentration into the percentage of intact viral capsid using a calibration curve with DNA concentration drawn for% of intact capsid standard.

Description

Technical Field

[0001] The present invention relates to interferometry-based biochemical assays. The present invention provides methods for determining DNA concentration in DNA viruses, such as adeno-associated viruses (AAV), and for determining empty versus complete viral capsids. Background Art

[0002] Adenovirus is a medium-sized non-enveloped virus with an icosahedral nucleocapsid containing a double-stranded DNA genome. Adenovirus particles consist of an icosahedral protein shell (capsid) surrounding a protein core containing a linear double-stranded DNA genome. The capsid, which is 70nm to 100nm in diameter, is composed of 252 structural capsomeres.

[0003] Herpes simplex virus type 1 (HSV-1) has a large linear double-stranded DNA genome in an icosahedral capsid shell, a cell-derived lipid envelope, and a proteinaceous inner membrane layer. More than fifty viral proteins and a variety of host proteins have been identified in the HSV-1 virion.

[0004] Adeno-associated viruses (AAVs) have become the vector of choice for gene therapy clinical trials because they are long-term expressed in humans and are non-pathogenic. 1. Wild-type AAVs consist of a single-stranded DNA genome of up to 4.8 kb in size, flanked by inverted terminal repeats (ITRs), and encapsidated in a protein capsid assembled from 60 proteins at a molar VP1:VP2:VP3 ratio of approximately 1:1:10.

[0005] In recent years, AAV vectors containing DNA transgenes encapsulated into protein capsids have shown great therapeutic potential. An inherent feature of the AAV manufacturing method is the production of capsids that are not encapsulated with therapeutic transgenes and are therefore called empty capsids. Typically, up to 95% of the capsids produced upstream of cell culture may be empty capsids, and this percentage has been shown to vary significantly between independent vector preparations. In its natural life cycle, wild-type AAV has also been shown to produce a large proportion of empty capsids. The clinical efficacy of these empty capsids is not yet fully understood, but it has been shown that there may be an enhanced immune response to high concentrations of viral particles and potential damage to efficacy through receptor competition. Empty capsids cannot provide the expected therapeutic benefits and are therefore considered to be product-related impurities. In addition to empty capsids, a heterogeneous population of partially filled (or intermediate) capsids may also be produced during the manufacturing process, which contains encapsulated process-related impurities or truncated genomes. Therefore, it is necessary to have an analytical technique that can provide information about the content distribution (called capsid content) of the AAV capsid.

[0006] Effective quantification of AAV titer and determination of DNA transgene content by empty / full (E / F) capsid ratio is a major challenge in the development and manufacture of gene therapy vectors. A range of currently available tools include AUC, TEM, ELISA, PCR-based methods, etc. TEM (transmission electron microscopy) and AUC (analytical ultracentrifugation) methods are based on capsid size differences (assuming that there is a correlation with DNA transgene content). qPCR is an established technique, but can take hours and requires development and optimization for each AAV vector. Digital droplet PCR (ddPCR) is more accurate than qPCR, but has a smaller dynamic range and requires precise sample dilution.

[0007] Current methods require purification of AAV samples prior to analysis. AAV purification methods are multi-step, labor-intensive, and time-consuming. Typical AAV purification methods include multiple steps involving filtration, column chromatography, and centrifugation. For example, commercial-scale AAV production includes the following steps: thawing HEK cells, amplifying HEK cells and transfecting, mechanical cell lysis (release of vector from cells), freezing, deep filtration (clarification), cross-flow filtration (concentration), iodixanol centrifugation (intact capsid separation), affinity chromatography, and filling and finishing. The complexity of the purification method is a major limitation to the accuracy, reproducibility, and adoption of E / F capsid measurements. In addition, purified samples are not necessarily very pure and may contain trace contaminants that can interfere with analytical methods.

[0008] It would be desirable to have analytical techniques that provide information about the distribution of the contents of viral capsids, referred to as capsid content. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1A One example of a biosensor interferometer system suitable for use with the disclosed methods is illustrated. Figure 1B and Figure 1C Each exemplifies a biosensor interferometer probe.

[0010] FIG. 2A to FIG. 2B It is shown that the first reflected light signal and the second reflected light signal form a spectral interference pattern.

[0011] Figure 3 The preparation of cross-linked Flow chart of (copolymer of sucrose and epichlorohydrin) 400-SPDP.

[0012] Figure 4 shows the preparation of streptavidin / HRP-crosslinked Flowchart of the conjugates.

[0013] Figure 5 shows the preparation of anti-fluorescein-cross-linked Flowchart of the process.

[0014] Figure 6 The steps for performing an assay for ssDNA are exemplified.

[0015] Figure 7 Shows the Figure 6 The illustrated protocol for the assay targeting ssDNA.

[0016] Figure 8 A standard curve for M13 plasmid DNA is presented. The wavelength shift corresponds to bound HRP enzyme activity.

[0017] Fig. 9 The wavelength shift (nm) versus time (seconds) for 24 repetitions in the BLI measurement is shown.

[0018] Fig.10 The 3 main steps of the ssDNA assay are outlined: AAV capture, lysis, and then total AAV DNA measurement.

[0019] Fig.11 A typical nm shift for capture of AAV at 4x1011 viral particles / ml is shown, which approaches plateau binding on the AAVX sensor after 30 minutes.

[0020] Fig.12 Results are plotted showing DNA concentration versus % of intact AAV capsids.

[0021] Fig.13 The steps for performing an assay for ssDNA using an anti-DNA-HRP conjugate are exemplified.

[0022] Fig.14 Shows the Fig.13 The illustrated protocol for the assay targeting ssDNA.

[0023] Fig.15 Presents the use Fig.14 The M13 plasmid DNA dose response scheme.

[0024] Fig.16 Shown are the wavelength shift (nm) versus time (sec) for AAV capture at 3 different AAV concentrations.

[0025] Fig.17 Shows Fig.16 ssDNA measurement results of captured AAV capsids (relationship between wavelength shift and time).

[0026] Fig.18 Shown is an AAV standard curve prepared from known standards.

[0027] Fig.19Shown are the results of ssDNA assays for two AAV samples by magnetic particle capture and BLI detection. DETAILED DESCRIPTION

[0028] definition

[0029] The terms used in the claims and specification should be interpreted according to their ordinary meanings as understood by those skilled in the art, except for the definitions set forth below.

[0030] As used herein, "about" means within ±10% of the stated value.

[0031] As used herein, an "analyte binding" molecule refers to any molecule that is capable of participating in a specific binding reaction with an analyte molecule. Examples include, but are not limited to: (i) an antigen molecule for detecting the presence of an antibody specific for the antigen; (ii) an antibody molecule for detecting the presence of an antigen; (iii) a protein molecule for detecting the presence of a binding partner for the protein; (iv) a ligand for detecting the presence of a binding partner; or (v) a single-stranded nucleic acid molecule for detecting the presence of a nucleic acid binding molecule.

[0032] The "aspect ratio" of a shape is the ratio of its longer dimension to its shorter dimension.

[0033] "Binding molecule" refers to a molecule that is capable of binding to another molecule of interest.

[0034] As used herein, "binding pair" refers to two molecules that attract each other and specifically bind to each other. Examples of binding pairs include, but are not limited to, antigens and antibodies to antigens, ligands and receptors thereof, complementary strands of nucleic acids, biotin and avidin, biotin and streptavidin, lectins and carbohydrates. Preferred binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, digoxin / anti-digoxin.

[0035] A "full AAV capsid" contains intact genomic material, whereas an "empty AAV capsid" contains no genomic material.

[0036] As used herein, "immobilization" refers to an agent being fixed to a solid surface. When an agent is immobilized to a solid surface, it is either non-covalently bound or covalently bound to the surface.

[0037] As used herein, "monolithic substrate" refers to a single piece of solid material having one refractive index, such as glass, quartz, or plastic.

[0038] As used herein, a BLI "probe" refers to a monolithic substrate having an aspect ratio (length to width) of at least 2 to 1 and coated with a thin film layer on the sensing side. The probe is typically made of a material suitable for BLI detection, such as glass. The probe has a distal end and a proximal end. The proximal end (also referred to as the probe tip in this application) has a sensing surface coated with a thin layer of analyte binding molecules.

[0039] "Waveguide" refers to a device (eg, a conduit, coaxial cable, or optical fiber) designed to confine and direct the propagation of electromagnetic waves (eg, light).

[0040] The present invention provides a method for determining the DNA concentration in a virus and determining the ratio of empty viral capsids to complete viral capsids. The initial step is to capture specific viruses by capturing a limited amount of capsid particles on a first solid surface. The first solid phase is designed for specific virus capture, wherein non-specific binding with interfering substances in the sample is negligible. The second step is to lyse the capsid to release viral DNA from the first solid phase into a lysis solution. After the lysis solution is separated from the first solid phase, the third step is to contact the lysis solution with a second solid surface. The second solid phase captures and measures the total amount of DNA derived from the captured capsid. Since the amount of capsid particles is known in the initial capture step and / or is consistent between samples, the total DNA assay signal is proportional to the E / F ratio.

[0041] In one method, biolayer interferometry (BLI) technology is used for detection. BLI is an optical technique for measuring macromolecular interactions by analyzing the interference pattern of white light reflected from the surface of the biosensor tip. The method is characterized by three basic steps to determine the DNA concentration in DNA viruses (such as AAV) and determine the ratio of empty capsids to complete capsids. The initial step is to perform specific virus capture on the tip of the first biosensor probe to a limited amount of capsid particles. The first probe is designed for specific virus capture, wherein the non-specific binding with interfering substances in the sample is negligible. The second step is to crack the capsid to release the viral DNA from the first probe into the lysis solution. After removing the first probe, the third step is to immerse the second biosensor probe tip in the lysis solution. The second biosensor probe tip measures the total DNA derived from the captured capsid. Since the amount of capsid particles is known in the initial capture step and is consistent between samples, the total DNA determination signal is proportional to the E / F ratio.

[0042] Biosensor Interferometer System

[0043] Figure 1A One example of a biosensor interferometer system suitable for use with the disclosed methods is illustrated. Figure 1ADepicted is a biosensor interferometer 100 (or simply "interferometer") that includes a light source 102, a detector 104, a waveguide 106, and an optical assembly 108 (also referred to as a "probe"). The probe 108 may be connected to the waveguide 106 via a coupling medium.

[0044] The light source 102 can emit white light that is guided by the waveguide 106 toward the probe 108. For example, the light source 102 can be a light emitting diode (LED) configured to generate light within a range of at least 50 nanometers (nm), 100 nm, or 150 nm within a specified spectrum (e.g., 400 nm or less to 700 nm or greater). Alternatively, the interferometer 100 can employ multiple light sources with different characteristic wavelengths, such as LEDs designed to emit light of different wavelengths in the visible range. The same functionality can be achieved with a single light source having appropriate filters for directing light of different wavelengths onto the probe 108.

[0045] The detector 104 is preferably a spectrometer, such as the Ocean Optics USB4000, which is capable of recording the spectrum of the interfering light received from the probe 108. Alternatively, if the light source 102 operates to direct different wavelengths onto the probe 108, the detector 104 can be a simple photodetector capable of recording the intensity at each wavelength. In another embodiment, the detector 104 can include multiple filters that allow detection of the intensity at each of the multiple wavelengths.

[0046] The waveguide 106 can be configured to transmit light emitted by the light source 102 to the probe 108, and then transmit light reflected by surfaces within the probe 108 to the detector 104. In some embodiments, the waveguide 106 is a bundle of optical fibers (e.g., a single-mode fiber optic cable), while in other embodiments, the waveguide 106 is a multi-mode fiber optic cable.

[0047] like Figure 1B As shown, the probe 108 includes a monolithic substrate 114, a thin film layer (also referred to as an "interference layer"), and a biomolecule layer (also referred to as a "biolayer") composed of analyte molecules 122, which have been bound to analyte binding molecules 120. The monolithic substrate 114 is composed of a transparent material through which light can pass. The interference layer is also composed of a transparent material. When light is irradiated on the probe 108, the proximal surface of the interference layer can act as a first reflective surface, and the biolayer can act as a second reflective surface. As further described below, the light reflected by the first reflective surface and the second reflective surface can form an interference pattern that can be monitored by the interferometer 100.

[0048] The interference layer typically includes multiple layers combined in a manner that improves the detectability of the interference pattern. For example, the interference layer is composed of a tantalum pentoxide (Ta2O5) layer 116 and a silicon dioxide (SiO2) layer 118. The tantalum pentoxide layer 116 can be thin (e.g., about 10nm to 40nm) because its main purpose is to improve the reflectivity at the near-side surface of the interference layer. At the same time, the silicon dioxide layer 118 can be relatively thick (e.g., about 650nm to 900nm) because its main purpose is to increase the distance between the first reflective surface and the second reflective surface.

[0049] To illustrate a simple interferometry test, a probe 108 can be suspended in a well 110 that includes a sample 112. During a diagnostic test, analyte molecules 122 will bind to analyte binding molecules 120 along the distal end of the probe 108, and these binding events will result in an interference pattern that can be observed by the detector 104. The interferometer 100 can monitor the thickness of the biological layer formed along the distal end of the probe 108 by detecting a shift in the phase characteristics of the interference pattern.

[0050] Figure 1C Another biosensor interferometer probe is illustrated. The probe includes a monolithic substrate, an interference layer coated on the second surface of the monolithic substrate, and an analyte binding molecule layer coated on the interference layer, wherein the monolithic substrate has a first surface and a second surface arranged substantially parallel to each other at the opposite ends of the monolithic substrate. The interference layer will usually be composed of magnesium fluoride (MgF2). When light is irradiated on the interference measurement sensor, the first interface between the monolithic substrate and the interference layer acts as a first reflection surface, and when light is irradiated on the probe, the second interface between the biological layer formed by the combination of the analyte molecules in the sample with the analyte binding molecules and the solution containing the sample acts as a second reflection surface. As described above, the thickness of the biological layer can be estimated based on the interference pattern of the light reflected by the first reflection surface and the second reflection surface.

[0051] The probe 200 includes an interference layer 204 fixed along the distal end of the monolithic substrate 202. Analyte binding molecules 206 can be deposited along the distal surface of the interference layer 204. During the biochemical test process, as analyte molecules 208 in the sample bind to the analyte binding molecules 206, a biolayer will be formed.

[0052] like Figure 1CAs shown, the monolithic substrate 202 has a proximal surface (also referred to as the "coupling side") that can be coupled to a waveguide of, for example, an interferometer, and a distal surface (also referred to as the "sensing side") on which additional layers are deposited. Typically, the monolithic substrate 202 has a length of at least 3 millimeters (mm), 5 mm, 10 mm, or 15 mm. In a preferred embodiment, the aspect ratio (length to width) of the monolithic substrate 202 is at least 5 to 1. In such embodiments, the monolithic substrate 202 can be said to have a cylindrical form. The cross-section of the monolithic substrate 202 can be circular, elliptical, square, rectangular, triangular, pentagonal, etc. The monolithic substrate 202 preferably has a refractive index that is substantially higher than the refractive index of the interference layer 204, so that the proximal surface of the interference layer 204 effectively reflects the light directed to the probe 200. The preferred refractive index of the monolithic substrate can be higher than 1.5, 1.8, or 2.0. Thus, the monolithic substrate 202 may be composed of a high refractive index material such as glass (refractive index 2.0) rather than a low refractive index material such as quartz (refractive index 1.46) or plastic (refractive index 1.32 to 1.49).

[0053] The interference layer 204 is composed of at least one transparent material, which is coated on the distal surface of the monolithic substrate 202. These transparent materials are deposited on the distal surface of the monolithic substrate 202 in the form of a thin film with a thickness ranging from a fraction of a nanometer (e.g., a monolayer) to several microns. The interference layer 204 can have a thickness of at least 500nm, 700nm, or 900nm. Exemplary thicknesses are between 500nm and 5,000nm (and preferably between 800nm ​​and 1,200nm). In this article, for example, the interference layer 204 has a thickness of approximately 900nm to 1,000nm or 940nm.

[0054] Compared to conventional probes, the interference layer 204 has a refractive index substantially similar to that of the biological layer. This ensures that the reflection from the distal end of the probe 200 is primarily due to the analyte molecules 208 rather than the interface between the interference layer 204 and the analyte binding molecules 206. In some embodiments, the interference layer 204 is composed of magnesium fluoride (MgF2), while in other embodiments, the interference layer 204 is composed of potassium fluoride (KF), lithium fluoride (LiF), sodium fluoride (NaF), lithium calcium aluminum fluoride (LiCaAlF6), sodium aluminum fluoride (Na3AlF6), strontium fluoride (SrF2), aluminum fluoride (AlF3), sulfur hexafluoride (SF6), etc. Magnesium fluoride has a refractive index of 1.38, which is substantially the same as the refractive index of the biological layer formed along the distal end of the probe 200. For comparison, the interference layer of a conventional probe is typically composed of silicon dioxide, and the refractive index of silicon dioxide is about 1.4 to 1.5 in the visible light range. Because the interference layer 204 and the biological layer have similar refractive indices, the light will experience minimal scattering as it travels from the interference layer 204 into the biological layer and then from the biological layer back into the interference layer 204 .

[0055] In one embodiment, probe 200 includes an adhesion layer, which is deposited along the distal surface of the interference layer 204 fixed to the monolithic substrate 202. The adhesion layer can be composed of a material that promotes the adhesion of the analyte binding molecule 206. An example of such material is silicon dioxide. Compared with the interference layer 204, the adhesion layer is usually very thin, so its impact on the light traveling toward the biolayer or returning from the biolayer will be minimal. For example, the adhesion layer 310 can have a thickness of about 3nm to 10nm, and the interference layer 304 can have a thickness of about 800nm ​​to 1,000nm. The biolayer formed by the analyte binding molecule 306 and the analyte molecule 308 will usually have a thickness of several nm.

[0056] When light is irradiated on probe 200, the near side surface of interference layer 204 can act as a first reflective surface, and the far side surface of biological layer can act as a second reflective surface. The presence, concentration or binding rate of analyte molecule 208 and probe 200 can be estimated based on the interference of the light beam reflected by these two reflective surfaces. When analyte molecule 208 is attached to (or detached from) analyte binding molecule 206, the distance between the first reflective surface and the second reflective surface will change. Since the size of all other components in probe 200 remains the same, the interference pattern formed by the light reflected by the first reflective surface and the second reflective surface is phase-shifted according to the change of the biological layer thickness caused by the binding event.

[0057] In operation, an incident light signal 210 emitted by a light source is transmitted toward the biological layer through the monolithic substrate 202. Within the probe 200, the light will be reflected at the first reflective surface, thereby generating a first reflected light signal 212. The light will also be reflected at the second reflective surface, thereby generating a second reflected light signal 214. The second reflective surface initially corresponds to the interface between the analyte binding molecules 206 and the sample in which the probe 200 is immersed. When binding occurs during a biochemical test, the second reflective surface becomes the interface between the analyte molecules 208 and the sample.

[0058] The first reflected light signal and the second reflected light signal form a spectral interference pattern, such as FIG. 2A to FIG. 2B When the analyte molecules bind to the analyte binding molecules on the far side surface of the interference layer, the optical path of the second reflected light signal will become longer. Figure 2B As shown, the spectral interference pattern shifts from T0 to T1. By measuring the phase shift after a period of time, the association rate of the analyte molecule with the analyte binding molecule fixed on the far side surface of the interference layer 204 can be used to calculate the analyte concentration in the sample. By measuring the phase shift continuously in real time, a kinetic binding curve can be drawn with the offset versus time. The association rate of the analyte molecule with the analyte binding molecule fixed on the far side surface of the interference layer can be used to calculate the analyte concentration in the sample. Therefore, the measurement of phase shift is the detection principle of thin film interferometer.

[0059] Method for determining DNA concentration in DNA viruses by biolayer interferometry

[0060] The present invention relates to methods for determining the DNA concentration in DNA viruses, such as adeno-associated virus (AAV), adenovirus and herpes simplex virus type 1 (HSV-1), by bio-layer interferometry.

[0061] First aspect

[0062] In a first aspect, the method comprises the following steps: (a) obtaining a first probe having a fixed amount of antiviral antibodies immobilized on the tip of the probe, wherein the virus is AAV, adenovirus or HSV-1; (b) dipping the tip of the first probe in a sample solution containing a virus sample to capture the virus on the probe under defined binding conditions; (c) dipping the first probe in a washing solution to wash the tip of the first probe; (d) dipping the first probe in a cleavage solution, and cleaving the virus from the tip of the first probe to obtain single-stranded DNA (ssDNA), and then removing the first probe from the cleavage solution; (e) dipping a second probe containing a first protein on the tip of the probe into the cleavage solution, wherein the first protein binds to the ssDNA; and (f) determining the ssDNA concentration in the sample by measuring the wavelength shift caused by light interference, and quantifying the wavelength shift against a calibration curve to determine the ssDNA concentration;

[0063] Second aspect

[0064] In a second aspect, the method is similar to the first aspect, except that after step (e), the method includes an additional step of amplifying the wavelength shift. In a second aspect, the method includes the following steps: (a) obtaining a first probe having a fixed amount of antiviral antibodies fixed to the tip of the probe, wherein the virus is AAV, adenovirus or HSV-1; (b) dipping the tip of the first probe in a sample solution containing a virus sample to capture the virus on the probe under defined binding conditions; (c) dipping the first probe in a washing solution to wash the tip of the first probe; (d) dipping the first probe in a lysis solution and cleaving the virus from the tip of the first probe to obtain single-stranded DNA (ssDNA), and then removing the first probe from the lysis solution; (e) dipping a second probe containing a first protein on the tip of the probe into the sample solution; and (f) dipping the first probe into the sample solution. (f) immersing the second probe in a reagent solution containing a biotin-labeled second protein, wherein the second protein binds to the ssDNA; (g) immersing the second probe in a conjugate solution containing a conjugate containing streptavidin and horseradish peroxidase (HRP) to allow the conjugate to bind to the second probe; (h) immersing the second probe in an HRP substrate solution to allow the substrate to bind to the HRP in the conjugate for a period of time; and (g) determining the ssDNA concentration in the sample by measuring a wavelength shift caused by light interference, and quantifying the wavelength shift against a calibration curve to determine the ssDNA concentration; Fig.10 One embodiment of the present invention is illustrated.

[0065] In step (a) and step (e) of the disclosed method, a second probe is obtained, the second probe having a tip for binding an analyte. The tip of the second probe for detecting ssDNA may have a small surface area of ​​≤5 mm, preferably ≤2 mm or ≤1 mm in diameter. The small surface of the probe tip provides several advantages. In solid phase immunoassays, probes with a small surface area have less nonspecific binding and therefore produce a lower background signal. In addition, due to the small surface area of ​​the tip, the reagent or sample carried on the probe tip is very small. This feature makes the probe tip easy to clean, and due to the large volume of the washing solution, the contamination in the washing solution is negligible. The negligible contamination of the washing solution and the small consumption of reagents enable the reagents and washing solutions to be reused multiple times, if necessary, for example, 3 to 10 times, 3 to 15 times or 3 to 20 times.

[0066] Methods of immobilizing proteins (such as antibodies) to a solid phase (the sensing surface of the probe tip) are common in immunochemistry. The protein can be directly bound to the solid phase by adsorption, or it can be indirectly bound to the solid phase by a binding pair. For example, the probe surface can be coated with a first member of the binding pair (e.g., an anti-hapten), and an anti-viral antibody labeled with a second member of the binding pair (e.g., a hapten) is immobilized on the probe by biotin-streptavidin binding.

[0067] In step (b) of the method, the first probe tip is immersed in a sample solution containing an AAV sample for a first period of time to capture a defined amount of virus on the probe under defined binding conditions. The binding time is from 10 minutes, 20 minutes or 30 minutes to 1 hour or 2 hours. For example, the binding time is from 10 minutes to 1 hour. In a preferred embodiment, the amount of antiviral on the probe is fixed and the binding conditions are fixed, capturing approximately the same amount of virus in different samples. Typically, the virus in the sample exceeds the maximum binding capacity of the antiviral on the probe. In one embodiment, the virus is allowed to bind close to the maximum binding of the antiviral on the probe.

[0068] This step provides an important aspect of the invention: the capture of AAV is normalized to the same number of viral particles from sample to sample.

[0069] In step (c), the first probe is immersed in a washing solution for a period of time (e.g., 5 seconds to 5 minutes, 10 seconds to 2 minutes, or 15 seconds to 1 minute), the washing solution comprising an aqueous solution having a pH preferably of 6.0 to 8.5. The aqueous solution can be water or a buffer having a pH of 6.0 to 8.5. Preferably, the aqueous solution contains 1 mM to 10 mM or 1 mM to 100 mM phosphate buffer, tris buffer, citrate buffer, or other buffer suitable for pH 6.0 to 8.5.

[0070] In step (d) of the method, the tip of the first probe is immersed in a lysis solution, and the lysis solution is heated to remove the DNA from the capsid, release the DNA into the lysis reagent, and denature the DNA to single strands. There are different methods for lysing DNA viruses and releasing DNA that can be used in the present invention; heat plus detergent lysis is preferred due to the speed. The lysis solution typically contains a detergent at a concentration of at least 1% or 2%. For example, the lysis solution contains 2% to 4% Tween 20. For ssDNA (e.g., AAV), the heating step is typically performed at a temperature of 50°C to 90°C, or 70°C to 90°C, or 80°C to 95°C, and for dsDNA, the heating step is typically performed at a temperature of 80°C to 95°C or 90°C to 100°C. The heating time is typically 2 minutes to 5 minutes or 2 minutes to 10 minutes. After cleaving the DNA from the first probe, the first probe is removed from the lysis solution.

[0071] In step (e), the second probe including the first protein on the probe tip is immersed in a lysis solution for a period of time (eg, 5 to 60 minutes), wherein the first protein binds to the ssDNA.

[0072] The second probe is then optionally washed in a wash solution as described in step (c).

[0073] In step (f), the second probe is immersed in a reagent solution containing a biotin-labeled second protein for a period of time (eg, 30 seconds to 5 minutes), wherein the second protein binds to the ssDNA.

[0074] The first protein and the second protein both bind to ssDNA, and they may be the same or different. For example, the first protein and the second protein are independently antibodies against ssDNA or single-stranded DNA binding proteins (SSB).

[0075] The second probe is then optionally washed in a wash solution as described in step (c).

[0076] In step (g), the second probe is immersed in a conjugate solution containing a polymer conjugate for a period of time (e.g., 10 seconds to 5 minutes, 15 seconds to 1 minute, or 20 seconds to 2 minutes) to allow the conjugate to bind to the second probe, wherein the polymer conjugate contains streptavidin and horseradish peroxidase (HRP).

[0077] Typically, the high molecular weight conjugate has a molecular weight of at least 200,000 Daltons, or at least 400,000 Daltons, or at least 500,000 Daltons, or at least 1 million Daltons, or at least 2 million Daltons. For example, the conjugate has a molecular weight of 200,000 Daltons to 10 million Daltons, 500,000 Daltons to 10 million Daltons, 1 million Daltons to 10 million Daltons, or 2 million Daltons to 10 million Daltons.

[0078] In one embodiment, the high molecular weight conjugate comprises streptavidin and cross-linked HRP, wherein the conjugate comprises at least 2, or at least 4, or at least 5, or at least 10 HRPs.

[0079] In one embodiment, the high molecular weight conjugate comprises streptavidin and HRP, wherein the streptavidin and HRP are cross-linked together. The conjugate comprises at least 2, or at least 4, at least 5, or at least 10 HRPs, and the molar ratio of streptavidin to HRP is between about 1:2 and about 2:1, or between about 1:1.5 and about 1.5 to 1, for example about 1:1.

[0080] In a preferred embodiment, the high molecular weight conjugate comprises streptavidin, HRP and a polymer. In the conjugate, streptavidin and HRP can be bound to a polymer. The polymer typically has a molecular weight of 200,000 to 10,000,000 Daltons, or 500,000 to 10,000,000 Daltons, or 1 million to 10 million Daltons, or 2 million to 10 million Daltons. The polymer can be a polysaccharide (e.g., dextran, amylose, polysucrose), a dendrimer or a polyethylene glycol. In a preferred embodiment, the polymer is (copolymer of sucrose and epichlorohydrin). The conjugate comprises at least 2, or at least 4, at least 5, or at least 10 HRPs, and the molar ratio of streptavidin to HRP is between about 1:2 to about 2:1, or between about 1:1.5 to about 1.5 to 1, for example about 1:1.

[0081] After binding, the second probe is then optionally washed 1 to 3 times in a washing solution as described in step (c).

[0082] In step (h), the second probe is immersed in an HRP substrate solution to allow the substrate to bind to the HRP in the conjugate for a period of time (eg, 30 seconds to 5 minutes).

[0083] When the product of the HRP / substrate reaction is bound to the surface of the optical layer, the optical layer increases its thickness and produces a nanometer wavelength shift due to light interference, and the wavelength shift is proportional to the amount of HRP on the probe. Any HRP substrate that binds the product to the solid surface is suitable for the present invention. Precipitated substrates are preferably used for BLI determination. Suitable substrates include 3,3'-diaminobenzidine tetrahydrochloride (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), benzamidine, 4-chloro-1-naphthol, chlorinated nitro tetrazolium blue and 5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt. Preferred substrates are chloronaphthol, benzamidine and tetramethylbenzidine, and more preferably chloronaphthol.

[0084] In step (i), the ssDNA concentration in the sample is determined by measuring the wavelength shift due to light interference, and the wavelength shift is quantified against a calibration curve to determine the ssDNA concentration. The phase shift can be monitored dynamically or determined by the difference between the start time point (T0) and the end time point (T1) (see Figure 2B ).

[0085] In the above-mentioned binding step and washing step, the reaction is optionally accelerated by stirring or mixing the solution in the container. For example, the flow of the solution across the probe tip, such as lateral flow or orbital flow, can be induced in one or more reaction containers (including sample containers, reagent containers, washing containers and conjugate containers) to accelerate the binding reaction and dissociation. For example, the reaction container can be mounted on an orbital oscillator, and the orbital oscillator rotates at a speed of at least 50rpm, preferably at least 200rpm or at least 500rpm, for example 50rpm to 200rpm or 500rpm to 1,500rpm. In addition, the probe tip can move up and down and perpendicular to the plane of the orbital flow at a speed of 0.01mm / second to 10mm / second to cause additional mixing of the solution above and below the probe tip.

[0086] In one embodiment, the second probe (anti-DNA probe) is immersed in the lysed sample only to bind DNA. It is not necessary to add reagents to the lysed sample. After the binding step, the anti-DNA probe is moved to another container to carry out the rest of the assay. The amount of DNA bound to the probe is a very small percentage of the DNA in the sample, so the composition and content of the sample are not changed. This produces the option of using the lysed sample for other analyses. Since the sample is affinity purified by the first antiviral probe, there is almost no protein residue in the purified sample, and it is easy to perform subsequent PCR. The user can choose to determine a specific sequence other than total DNA.

[0087] The third aspect

[0088] In a third aspect, the method is similar to the method of the second aspect, except that the second probe is immersed in a reagent solution containing an HRP-labeled second protein that binds to ssDNA.

[0089] In a third aspect, a method for determining the concentration of DNA in a virus comprises the following steps: (a) obtaining a first probe having a fixed amount of antiviral antibodies fixed to the tip of the probe, wherein the virus is AAV, adenovirus or HSV-1; (b) dipping the tip of the first probe in a sample solution containing a virus sample to capture the virus on the probe under defined binding conditions; (c) dipping the first probe in a washing solution to wash the tip of the first probe; (d) dipping the first probe in a lysis solution and cleaving the virus from the tip of the first probe to obtain single-stranded DNA (ssDNA), and then removing the first probe from the lysis solution; (e) (f) immersing a second probe comprising a first protein on the tip of the probe in the lysis solution, wherein the first protein binds to ssDNA; (g) immersing the second probe in a reagent solution comprising a second protein labeled with HRP, wherein the diameter of the tip surface of the second probe is ≤5 mm, and the second protein binds to ssDNA; (h) immersing the second probe in an HRP substrate solution to allow the substrate to bind to the HRP bound to the second probe for a period of time; and (h) determining the ssDNA concentration in the sample by measuring a wavelength shift caused by light interference, and quantifying the wavelength shift against a calibration curve to determine the ssDNA concentration.

[0090] In this method, the first protein and the second protein are independently antibodies against ssDNA or single-stranded DNA binding proteins (SSBs).

[0091] In the second aspect of the method, steps (a) to (e) are the same as those described in the first aspect.

[0092] In step (f), the second probe is immersed in a conjugate solution containing the HRP-labeled second protein that binds to DNA for a period of time (e.g., 10 seconds to 5 minutes, 15 seconds to 1 minute, or 20 seconds to 2 minutes) to allow the HRP-second protein conjugate to bind to the second probe.

[0093] After binding, the second probe is then optionally washed 1 to 3 times in a washing solution as described in step (c).

[0094] In step (g), the second probe is immersed in an HRP substrate solution to allow the substrate to bind to the HRP bound to the second probe for a period of time (eg, 30 seconds to 5 minutes). The HRP substrate is described in the first aspect of the method.

[0095] In step (h), the ssDNA concentration in the sample is determined by measuring the wavelength shift due to light interference as described in the first aspect of the method.

[0096] The common reagents and common steps in the first and second aspects of the method are similar.

[0097] Method for determining empty versus complete viral capsids by biolayer interferometry

[0098] The present invention relates to a method for measuring the percentage of intact viral (e.g., AAV) capsids in a viral (e.g., AAV) sample; also known as measuring empty (empty viral capsids) versus intact (intact viral capsids).

[0099] First aspect

[0100] The BLI signal of a captured DNA virus (e.g., AAV) is proportional to the number of capsids captured in real time. In theory, if the standard and sample have the same virus (e.g., AAV) concentration or the same number of captured capsids, the BLI signal of virus binding is the same, and the ssDNA signal is proportional to the empty capsid / complete capsid ratio, and the empty capsid / complete capsid ratio of a virus (e.g., AAV) sample can be determined from a standard curve, where the calibrator contains a mixture of empty capsids and complete capsids at different ratios. The calibrators are run at the same virus concentration.

[0101] In a first aspect, the method comprises first determining the ssDNA concentration in a viral sample according to the method described above, and then converting the ssDNA concentration to % of intact viral capsids using a calibration curve with DNA concentration plotted against % of intact viral capsid standards. The calibration curve can be established using the results of calibrators run with unknown samples, or using the results from calibrators run separately.

[0102] The present invention is an easy and accurate method for determining the empty / complete ratio of viral (e.g., AAV) transgenes. The method uses two probes (antiviral probe and anti-ssDNA probe) and includes three key steps: viral capture, lysis and total ssDNA quantification. The anti-AAV BLI probe of the present invention demonstrates reproducible and high-precision AAV capture, which can be monitored in real time. The binding of AAV to the anti-AAV sensor can be detected by BLI without the use of a label.

[0103] It is convenient to perform both the AAV capture step and the ssDNA quantification step with a BLI instrument and an automated protocol.

[0104] The second aspect (normalization)

[0105] If the standards and samples have different virus concentrations or different numbers of captured capsids, the BLI signals for virus (e.g., AAV) binding can be different; which can then bias the data analysis in the ssDNA assay to derive the empty / intact ratio in the virus sample. The present invention provides methods for normalizing the BLI signals for virus binding and aligning sample concentration differences to determine the empty / intact capsid ratio.

[0106] When using normalization to determine the empty capsid / complete capsid ratio, the virus binding signal in each sample is normalized by comparing it with the virus binding signal of one or more calibrants to give a normalization factor. The ssDNA signal (wavelength) of the sample is multiplied by the normalization factor to produce a normalized wavelength shift value for the sample. The calibrants are run at the same virus concentration and used to generate a standard curve of the relationship between the BLI signal and the empty virus capsid / complete virus capsid ratio. The DNA virus sample is run with the calibrant, and the wavelength shift value of the sample is normalized by the normalization factor, and then the empty / complete ratio of the sample is determined from the standard curve. Since the normalized wavelength shift value takes into account the capture differences caused by different virus concentrations in the sample, in some cases, normalization can be more accurate to calculate the empty / complete ratio.

[0107] In a first embodiment, the method comprises the following steps: (a) obtaining a first probe having a fixed amount of anti-DNA virus (e.g., AAV) antibody immobilized on the tip of the probe; (b) immersing the tip of the first probe in a sample solution containing a DNA virus sample to capture the AAV on the probe for a period of time under defined binding conditions, thereby measuring a first wavelength shift due to light interference caused by the bound virus on the probe; (c) determining a normalization factor based on a comparison of the first wavelength shift of the sample with (i) the first wavelength shift of a calibrator or (ii) the average of the first wavelength shifts of all calibrants; (d) immersing the first probe in a washing solution to wash the first probe tip; (e) immersing the first probe in a lysis solution, and cleaving the AAV from the tip of the first probe to obtain single-stranded DNA (ssDNA), and then removing the first probe from the lysis solution; and (f) removing the first probe from the lysed solution at the tip of the probe. A second probe comprising a first protein on the needle tip is immersed in the lysis solution, wherein the first protein binds to ssDNA; (g) the second probe is immersed in a reagent solution comprising a biotin-labeled second protein, wherein the second protein binds to ssDNA; (h) the second probe is immersed in a conjugate solution comprising a conjugate containing streptavidin and horseradish peroxidase (HRP) to allow the conjugate to bind to the second probe; (i) the second probe is immersed in an HRP substrate solution to allow the substrate to bind to the HRP in the conjugate for a period of time; and the second wavelength shift caused by light interference is measured; (i) the normalization factor is applied to the second wavelength shift to generate a normalized wavelength shift; and (k) the normalized wavelength shift is quantified against a calibration curve with a normalized wavelength shift drawn for the % of the complete AAV capsid to determine the percentage of the complete AAV capsid in the sample. The first protein and the second protein are independently SSB or anti-DNA antibodies. The first protein and the second protein can be the same or different.

[0108] In this method, details of steps (a), (b), (d) to (i) are similar to the corresponding steps in the above-mentioned method for determining the DNA concentration.

[0109] In step (c), the normalization factor is determined by comparing the first wavelength shift of the virus bound to the probe of the sample with the first wavelength shift of one or more calibrators. The calibrator is a virus (e.g., AAV) sample having a known empty capsid / complete capsid ratio and captured at the same viral concentration as other calibrators. The calibrator can be run with the sample under the same assay conditions. Alternatively, the calibrator can be run separately from the sample under the same assay conditions. Typically, 1 to 6, 2 to 6, 3 to 6, 2 to 5 or 3 to 5 calibrators are used to establish a normalization factor. The normalization factor is determined by the ratio of the first wavelength shift of the sample to the first wavelength shift of (i) calibrators or (ii) the average value of the first wavelength shift of all calibrators. In a preferred embodiment, the normalization factor is determined by the ratio of the first wavelength shift of the sample to the average value of the first wavelength shift of all calibrators.

[0110] In step (i), a normalized wavelength shift of the second wavelength shift is calculated by applying a normalizing factor to the second wavelength shift.

[0111] In step (i), the normalized wavelength shift is quantified by reference to a calibration curve to determine the percentage of intact viral capsids in the sample. A calibration curve is established by measuring the normalized shift of calibrator samples having a known percentage of intact viral capsids and plotting the normalized wavelength shift for the percentage of intact viral capsids.

[0112] Typically, samples and calibrants are run in the same run, but they can be run in different runs on the same day. If the samples and instrument are stable, the calibrants can be run on another day.

[0113] The normalization procedure for this embodiment is exemplified in Example 12.

[0114] In a second embodiment, the method is similar to the method of the first embodiment, except that the signal detection is free of biotin-streptavidin amplification. In a second embodiment, the method includes the following steps: (a) obtaining a first probe having a fixed amount of anti-DNA virus (e.g., AAV) antibodies fixed to the tip of the probe; (b) immersing the tip of the first probe in a sample solution containing a DNA virus sample to capture the AAV on the probe for a period of time under defined binding conditions, thereby measuring a first wavelength shift due to light interference caused by the bound virus on the probe; (c) determining a normalization factor based on a comparison of the first wavelength shift of the sample with (i) the first wavelength shift of the calibrant or (ii) the average of the first wavelength shifts of all calibrants; (d) immersing the first probe in a washing solution to wash the first probe tip; (e) immersing the first probe in a cleavage solution and cleaving the AAV from the tip of the first probe to obtain single-stranded DNA (ssDNA). A), then removing the first probe from the lysis solution; (f) immersing a second probe comprising a first protein on the probe tip in the lysis solution, wherein the first protein binds to ssDNA; (g) immersing the second probe in a reagent solution comprising an HRP-labeled second protein, wherein the second protein binds to ssDNA; (h) immersing the second probe in an HRP substrate solution to allow the substrate to bind to the HRP bound to the second probe for a period of time, and measuring the second wavelength shift caused by light interference; (i) applying the normalization factor to the second wavelength shift to generate a normalized second wavelength shift; and (ii) quantifying the normalized second wavelength shift against a calibration curve with normalized wavelength shifts plotted against the % of intact AAV capsids to determine the percentage of intact AAV capsids in the sample.

[0115] High molecular weight HRP conjugate

[0116] The present disclosure also provides a method comprising streptavidin, HRP and cross-linking Conjugates. Cross-linking The conjugate has a molecular weight of at least 500,000 Daltons, and preferably at least 1 million Daltons, or 2 million Daltons, or 5 million Daltons, or 10 million Daltons. The conjugate has at least 2 streptavidins and at least 2 HRPs, and preferably has at least 4 streptavidins and at least 4 HRPs. In one embodiment, the cross-linked having a molecular weight of 500,000 Daltons to 10 million Daltons or 1 million Daltons to 10 million Daltons, and each cross-linked Combine at least 2 HRPs and at least 2 streptavidins. In one embodiment, cross-linking having a molecular weight of at least 2 million Daltons (e.g., 1 million Daltons to 10 million Daltons), and each cross-linked Combine at least 4 or 5 HRP and at least 4 or 5 streptavidin. In one embodiment, cross-linking having a molecular weight of at least 2 million Daltons or at least 5 million Daltons (e.g., 2 million Daltons to 10 million Daltons or 5 million Daltons to 10 million Daltons), and each cross-linked Combine at least 10 HRP and at least 10 streptavidin.

[0117] In one embodiment, the conjugate has a ratio of 1:2-10:2-10 In another embodiment, the conjugate has a molar ratio of streptavidin:HRP of 1:3-7:3-7. Molar ratio of streptavidin:HRP. For example, The molecule binds at least 4 to 10 HRP and 4 to 10 streptavidin.

[0118] BLI probes

[0119] The present invention also provides a probe, which includes a single-chip substrate, a thin film layer and a biomolecule layer fixed on the tip of the probe, wherein the biomolecule layer includes analyte molecules, analyte binding molecules, HRP and chloronaphthol, wherein the diameter of the tip surface is ≤5mm.

[0120] Methods for determining DNA concentration in DNA viruses by different solid phase and non-BLI immunoassays

[0121] The present invention also relates to methods for determining DNA concentration in DNA viruses such as adeno-associated virus (AAV), adenovirus, and herpes simplex virus type 1 (HSV-1) by non-BLI solid phase immunoassay.

[0122] The method comprises the following steps: (a) obtaining a first solid surface immobilized with a fixed amount of anti-viral antibodies, wherein the virus is AAV, adenovirus or HSV-1; (b) contacting the first solid surface with a sample solution containing a virus sample to capture the virus on the first solid surface under defined binding conditions; (c) washing the first solid surface with a washing solution to remove unbound materials from the solid surface; (d) contacting the first solid surface with a lysis solution, and lysing the virus from the first solid surface to obtain single-stranded DNA (ssDNA) in the lysis solution, and then collecting the lysis solution; (e) contacting the ssDNA-containing lysis solution with a second solid surface immobilized with a first protein, wherein the first protein binds to the ssDNA; (f) washing the second solid surface; (g) contacting the second solid surface with a second protein conjugated with a label; and (h) detecting the label and determining the DNA concentration in the virus; wherein the first protein and the second protein are independently SSB or anti-DNA antibodies. The first protein and the second protein can be the same or different. The label can be an enzyme, a fluorescent label (e.g., Cy3, Cy5, Alexa Fluor TM ) or radioactive isotopes.

[0123] In one embodiment, the above step (g) can be replaced by step (g1) and step (g2) to amplify the signal; wherein (g1) is: contacting the second solid surface with a second protein conjugated with biotin, and (g2) is: contacting the second solid surface with a conjugate comprising streptavidin and a label for detection.

[0124] In one embodiment, the anti-viral antibodies are coated on the first solid surface by first immobilizing streptavidin on the solid surface and then incubating the solid surface with a biotin-anti-viral conjugate.

[0125] The first solid surface and the second solid surface can be independently selected from: beads, such as magnetic beads; tubes; microwells, such as 96-well or 384-well microplates containing ELISA formats; micron and nanometer-sized polystyrene beads or latex beads; nanospheres; microspheres; Sepharose, agarose, dextran and other carbohydrate-based resins; glass / silica, borate silicate / quartz materials (in the form of slides, needles, fibers, microspheres or beads); magnetic particles having latex, dextran, a polymer matrix in the form of nano, microspheres or rods; quantum dot nanocrystals, europium chelate conjugates (with or without a polymer coating on the europium surface).

[0126] The first solid surface and the second solid surface may be made of the same material or different materials.

[0127] In one embodiment, the first solid phase and / or the second solid phase are magnetic beads.

[0128] Method for determining DNA concentration in DNA viruses using two different solid surfaces and BLI for detection Law

[0129] The present invention also relates to a method for determining the DNA concentration in DNA viruses, such as adeno-associated virus (AAV), adenovirus, and herpes simplex virus type 1 (HSV-1), using two different solid surfaces for capturing viral capsids and BLI and ssDNA for detection.

[0130] The method comprises the following steps: (a) obtaining a first solid surface immobilized with a fixed amount of anti-viral antibodies, wherein the virus is AAV, adenovirus or HSV-1; (b) contacting the first solid surface with a sample solution containing a virus sample to capture the virus on the first solid surface under defined binding conditions; (c) washing the first solid surface with a washing solution to remove unbound materials from the solid surface; (d) contacting the first solid surface with a lysis solution, and lysing the virus from the first solid surface to obtain single-stranded DNA (ssDNA) in the lysis solution, and then collecting the lysis solution; (e) soaking the probe in water and immersing the probe in water; (i) determining the ssDNA concentration in the sample by measuring the wavelength shift caused by light interference, and quantifying the wavelength shift against a calibration curve to determine the ssDNA concentration.

[0131] In one embodiment, the above step (g) can be replaced by step (g1) and step (g2) to amplify the signal; wherein (g1) is: immersing the probe in a solution containing a second protein conjugated to biotin, and (g2) is: immersing the probe in a solution containing a conjugate containing streptavidin and HRP.

[0132] In one embodiment, the anti-viral antibodies are coated on the first solid surface by first immobilizing streptavidin on the solid surface and then incubating the solid surface with a biotin-anti-viral conjugate.

[0133] The first solid surface can be selected from: beads, such as magnetic beads; tubes; microwells, such as 96-well or 384-well microplates containing ELISA formats; micron and nano-sized polystyrene beads or latex beads; nanospheres; microspheres; Sepharose, agarose, dextran and other carbohydrate-based resins; glass / silica, borate silicate / quartz materials (in the form of slides, needles, fibers, microspheres or beads); magnetic particles having latex, dextran, a polymer matrix in the form of nano, microspheres or rods; quantum dot nanocrystals, europium chelate conjugates (with or without a polymer coating on the europium surface).

[0134] In one embodiment, the first solid phase is magnetic beads. The magnetic beads have a large surface area to capture viral capsids.

[0135] In one embodiment, the probe is a glass probe having a tip surface of ≤ 5 mm.

[0136] The present invention is further illustrated by the following examples, which are not to be construed as limiting the scope of the invention to the specific procedures described therein.

[0137] Example

[0138] Materials and instruments used in Examples 1 to 12

[0139] Recombinant streptavidin was obtained from IBA. Horseradish peroxidase (HRP) RZ=1 was obtained from Santa Cruz BioTech. SMCC, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester, DMF, anhydrous dimethylformamide were obtained from Thermo Scientific without further purification before use. Cy5-NHS, cyanine 5 monosuccinimidyl ester was ordered from ATT Bioquest. N-acetyl-L-cysteine ​​and n-ethylmaleimide were purchased from MP Biomedicals and Thermo Scientific. 10x phosphate buffered saline (ThermoScientific) was used by diluting 10 times with MilliQ water before use. 0.5M carbonate buffer, pH 9.4, was used directly from BioWorld supply.

[0140] AAVX probe (Gator Bio, Inc.) was used to capture AAV.

[0141] UV / VIS measurements were performed by spectrophotometer (Thermo BioMate 3S). Centrifugation was performed by centrifuge (Beckman Allegra 6R). Protein was desalted by Zeba spin column 2 ml size, 7K MWCO (Thermo Scientific). Bioconjugates were purified by custom packed Cytiva CL-4BSepharose medium in XK 16x400 columns connected to Cytiva AKTA Go purification system.

[0142] Will The Prime system (Gator Bio, Inc.) was used for BLI measurements.

[0143] Materials and Reagents Used in Examples 13 to 17 (Non-BLI Methods)

[0144] Dynabeads MyOne Streptavidin C1, catalog number 65001, 65002

[0145] Single-stranded DNA binding protein (SSB), ThermoFisher Scientific, catalog number 70032Z500UG

[0146] M13 single-stranded DNA, New England Biolabs, catalog number N4040S

[0147] Anti-ssDNA / dsDNA antibody, Absolute Antibody, catalog number Ab00347-1.1

[0148] Anti-ssDNA / dsDNA-HRP conjugate, Absolute Antibody, catalog number Ab00347-1.1-HRP

[0149] Empty AAV8 capsid, Vigene BioSciences, catalog number AV8-ET

[0150] Complete AAV8 capsid, Vigene Biosciences, catalog number CV10008

[0151] EZLabelTM Protein FITC Labeling Kit, BioVision, catalog number K832-5

[0152] Tween-20, Sigma, catalog number P1379-100ML

[0153] TMB substrate, Thermofisher Scientific, catalog number 34022

[0154] Stop solution, 0.3M H2SO4

[0155] Thermo Scientific TM Pierce TM 96-well polystyrene white opaque microplate, FisherScientific, catalog number 15042

[0156] Example 1. Cross-linking Preparation of 400-SPDP

[0157] According to Example 1 of US2001 / 0312105, cross-linked 400-SPDP (6-[3-[2-pyridyldithio]-propionylamino]hexanoic acid succinimidyl ester, Invitrogen). Figure 3 A flow chart for its preparation is shown.

[0158] Example 2. Preparation of cyanine 5 and maleimide-labeled streptavidin

[0159] Dissolve 10 mg of streptavidin in 500 μL PBS buffer to 20 mg / ml. Add 50 μL of 0.5 M carbonate buffer to the streptavidin solution. Add 5 mg / ml cyanine 5 monosuccinimidyl ester in 30 μL of anhydrous dimethylformamide (DMF) (MCR=1) to the streptavidin solution and allow it to react at room temperature for 1 hour. In this case, Cy5 is used as a tracer to enable the subsequent preparation of Cy5-streptavidin / HRP-crosslinked The streptavidin content in the conjugate is quantified. 10 mg / ml SMCC, 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester was freshly prepared in anhydrous dimethylformamide (DMF), and 52 μL (MCR=8.7) thereof was added to the above Cy5-labeled streptavidin. The SMCC labeling reaction was run for another hour at room temperature. A 2 ml size Zeba desalting spin column (7K MWCO) was prepared according to the ThermoScientific instruction guide. The Cy5 / SMCC dual-labeled streptavidin reaction mixture was loaded onto the column and centrifuged at 1000 x g for 2 minutes, and the flow-through containing the sample was collected. The concentration of streptavidin and the Cy5 labeling ratio were estimated by spectrophotometer at 280 nm and 655 nm absorption. Streptavidin was labeled with Cy5 so that labeled streptavidin could be monitored throughout the conjugation step.

[0160] Example 3. Preparation of maleimide-labeled horseradish peroxidase

[0161] 12mg horseradish peroxidase (HRP) is dissolved in 600μL 1xPBS buffer, followed by 60μL 0.5M carbonate buffer. 72μL freshly prepared 10mg / ml SMCC, 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester in anhydrous dimethylformamide (DMF) is added to the HRP solution to make the molar ratio of SMCC to HRP be MCR=7.2. After 1 hour of reaction at room temperature, 2ml Zeba desalting centrifugal column (7K MWCO) is prepared according to the Thermo Scientific instruction guide. The reaction mixture of maleimide-labeled HRP is loaded onto the top of the centrifugal column and centrifuged for 2 minutes at 1000x g. Purified samples are collected and the amount of maleimide-labeled HRP is estimated by spectrophotometer with 280nm absorption.

[0162] Example 4. Cy5-streptavidin, horseradish peroxidase, cross-linking Preparation of conjugates

[0163] Streptavidin / HRP crosslinking The flow chart is as follows Figure 4 Depicted. Cross-linking -SPDP, (3-(2-pyridyldithio) propionic acid succinimidyl ester) and DTT (dithiothreitol) 38mg / ml were reacted at room temperature for 1 hour at a 1:500 molar excess. At the end of the reaction, the mixture was loaded onto a 2ml size Zebra desalting spin column (7K MWCO) prepared according to the Thermo Scientific instruction manual. The thiol-labeled cross-linked peptide was collected as the flow-through of the Zeba spin column by centrifugation at 1000x g for 2 minutes. The sample was reacted with cyanine 5 / maleimide-labeled streptavidin (Example 2) and maleimide-labeled horseradish peroxidase (Example 3) in Table 1.

[0164] Table 1. Initial ratios of reagents in the reaction mixture

[0165]

[0166] Crosslink The coupling of thiol (1 mg) with maleimide-streptavidin (1.1 mg) and maleimide-horseradish peroxidase (0.8 mg) was carried out at room temperature for 4 hours, and then quenched with n-acetyl-1-cysteine ​​16 mg / ml (6.25 μL per mg streptavidin) and n-ethylmaleimide 32 mg / ml (10 μL per mg streptavidin) in 1xPBS. The quenched reaction mixture was purified with a CL-4B column with an AKTA Go purification system at a flow rate of 1 ml / min (1xPBS mobile phase). The first elution peak is the desired product.

[0167] Example 5. Anti-fluorescein-crosslinking Preparation of conjugates

[0168] Figure 5 Representative for the preparation of anti-fluorescein-cross-linked 1.5 mg / ml anti-fluorescein (Jackson ImmunoResearch) in 1 ml PBS was mixed with 1.9 μL 5 mg / ml SMCC in DMF and reacted at room temperature for 1 hour, followed by purification on a PD 10 column.

[0169] Cross-linking The thiol on -SPDP was deprotected by adding 30 μL of 38 mg / ml DTT to 0.7 mg of cross-linked 400-SPDP and reacted at room temperature for 1 hour, then the cross-linked Cross-linking of anti-fluorescein-SMCC -SH were mixed and reacted at room temperature overnight. Then 10 μL of 12.5 mg / ml NEM (Aldrich) was added and reacted at room temperature for 1 / 2 hour. The conjugate was then purified on a Sepharose 4B CL column.

[0170] Example 6. Preparation of reagents and anti-ssDNA probes for ssDNA assay

[0171] A quartz probe with a diameter of 1 mm and a length of 2 cm with a BLI optical layer at the distal tip was coated with aminopropylsilane using a chemical vapor deposition method (Yield Engineering Systems, 1224P) according to the manufacturer's protocol. The probe tip was then immersed in 10 μg / ml of mouse monoclonal anti-fluorescein-cross-linked (from Example 5) in PBS (pH 7.4). After allowing the antibody to adsorb to the probe for 15 minutes, the probe tip was washed in PBS.

[0172] The probe tip was then immersed in a solution containing 10 μg / ml of fluorescein-labeled mouse monoclonal anti-ssDNA (Sigma catalog number MAB3868). The antibody was fluorescein labeled by standard methods. After 10 minutes, the probe was washed in PBS. The probe was then immersed in a 15% sucrose solution and then dried at 37°C for 20 minutes. The monoclonal anti-ssDNA (Sigma catalog number MAB3868) was biotinylated by standard methods.

[0173] Example 7A: ssDNA Assay

[0174] Figure 6 The steps for performing an assay for ssDNA are illustrated. The anti-ssDNA probe (Example 6) is immersed in the ssDNA sample, followed by washing, and then immersed in B-anti-ssDNA reagent, followed by washing, and then immersed in streptavidin / HRP- The probe was then immersed in a substrate, followed by washing. Finally, the probe was immersed in a substrate, where the bound HRP enzyme activity was monitored by a BLI instrument (Gator Prime, GatorBio) according to the manufacturer's protocol. A chloronaphthol substrate (1.68 mM) was used. When chloronaphthol binds to HRP, the optical layer increases its thickness, resulting in a nanometer shift proportional to the amount of HRP on the probe.

[0175] Figure 7 Detailed information for performing the ssDNA assay, ie, reagent concentrations, volumes, etc., is shown. Figure 8 The samples with M13 plasmid DNA (New England Biolabs, catalog number N4040S) are presented (see Figure 7 The nanometer shift of the standard curve of step 12) corresponds to the bound HRP enzyme activity. This ssDNA assay uses a monoclonal anti-ssDNA antibody, however, other ssDNA binding proteins such as SSB (single-stranded DNA binding protein) can also be used.

[0176] Example 7B. ssDNA Assay Using Different Streptavidin / HRP Conjugates

[0177] Used as in Example 7A and Figure 7 The ssDNA assay was performed using the same protocol as described in , except that the streptavidin (SA) / HRP reagent used was: (i) SA / HRP-cross-linked (ii) HRP-SA conjugate (ThermoFisher, Ultra Streptavidin-HRP, N504, 1:1500), or (iii) Streptavidin-HRP (Sigma-Aldrich, Streptavidin-Peroxidase Polymer, S2438, 1:1000 dilution).

[0178] The results for the different conjugates are shown in Table 2. SA / HRP- The specific DNA signal of the conjugate was better.

[0179] Table 2. DNA signals of different conjugates

[0180]

[0181] Example 8: Reproducibility of AAV binding

[0182] AAVX probes (AAVX biosensors) coated with anti-AAV (GatorBio #PL168-160017) were immersed in 200 μL of sample containing 5×10 10 Binding occurred at 1000 orbital rpm while nm shift was monitored in a Gator Prime BLI instrument. Fig. 9 Wavelength shift (nm) versus time (seconds) for 24 replicate probe measurements is shown. Binding was highly reproducible from probe to probe with a coefficient of variation of 3%.

[0183] Example 9: Empty and Full (E / F) Method

[0184] Fig.10 The three main steps of the invention are summarized: AAV capture, lysis, and then total AAV DNA measurement. The AAV capture step uses the AAVX biosensor to specifically capture AAV in the sample. AAVX has a camelid nanobody (CapSelect TM , ThermoFisher) coated probe tip. Capture is allowed to proceed until the maximum binding capacity of the probe is met. After the capsid capture and wash sequence, the probe is transferred to a microwell containing a lysis reagent to release DNA from the capsid. After the lysis step, the AAVX probe is removed and the assay for ssDNA is performed as described in Example 1. Since the same number of capsids are initially captured, the ssDNA concentration in the lysis sample is proportional to the E / F ratio.

[0185] In this example, a 200 μL sample of a mixture of empty and complete AAV capsids was bound to the AAVX biosensor in a Gator Prime instrument at 1000 orbital rpm for 1 / 2 hour. Fig.11 Shown in 4×10 11 The typical nm shift of captured AAV at 100 viral particles / ml approached plateau binding on the AAVX sensor after 30 minutes as monitored by GatorPrime.

[0186] Empty capsids and mixtures of 0%, 25%, 50% and 75% intact intact capsids in PBS were captured by AAVX and produced similar and consistent nm shifts of 20.0, 21.8, 22.2 and 22.5, respectively. This indicates that the capture of different AAV samples in the first step was consistent. The consistent nm shift indicates that comparable amounts of capsids were captured by the probe in the AAV samples. After a wash step in 200 μL PBS, the probe was then immersed in 200 μL lysis reagent (PBS, pH 7.4, 3% Tween 20) and placed in a laboratory heating block at 90°C for 5 minutes, followed by cooling to room temperature for 2 minutes. The AAVX probe was removed and the sample containing the capsid DNA was placed back into the Prime instrument and the assay for ssDNA as described in Example 7 was performed. Fig.12 Results are depicted for DNA levels at various % intact AAV capsids. DNA levels correlated with % intact capsids, R 2 is 0.985.

[0187] Example 10: Preparation of SSB probe

[0188] A quartz probe with a diameter of 1 mm and a length of 2 cm with a BLI optical layer at the distal tip was coated with aminopropylsilane using a chemical vapor deposition method (Yield Engineering Systems, 1224P) according to the manufacturer's protocol. Figure 1B and Figure 1C The BLI probe is shown. The probe tip was immersed in a 10 μg / ml solution of mouse monoclonal anti-fluorescein-cross-linked FICOLL (from Example 5) in PBS (pH 7.4). After allowing the antibody to adsorb to the probe for 15 minutes, the probe tip was washed in PBS.

[0189] The probe tip was then immersed in a solution containing fluorescein-labeled SSB (ThermoFisher: 70032Z500UG). SSB was fluorescein-labeled by standard methods. The probe tip was immersed in a 10 μg / ml fluorescein-labeled SSB solution. After 10 minutes, the probe was washed with PBS and then dried at 37°C for 20 minutes.

[0190] Example 11: DNA Assay Using SSB-Coated Probes

[0191] Fig.13 The SSB probe assay format is shown. Fig.14 The assay protocol is listed, and Fig.15 M13 plasmid DNA dose responses are presented.

[0192] Example 12: Normalization method

[0193] In the following example, it is demonstrated how the normalization method can be used to calculate the empty / full ratio of AAV samples diluted to different concentrations.

[0194] AAV8 samples (approximately 50% intact ratio) were diluted with PBS to produce three concentrations at 2E11 vp / ml, 8E10 vp / ml, and 3E11 vp / ml. Since only PBS was used for dilution, these samples contained the same empty / intact ratio but different concentrations. Capture of capsids was performed in duplicate using the AAVX probe at these different AAV concentrations. PBS was used as a control. The corresponding binding curves and data are shown below. Since the BLI capture signal increases with AAV concentration, Fig.16 The binding data in showed wavelength shifts in the following order as expected: 2E11 vp / ml > 8E10 vp / ml > 3E10 vp / ml.

[0195] By using the software, we extracted Fig.16 The endpoints of each binding curve in the , and the replicate BLI data were averaged, as shown in Table 3. To generate the corresponding normalization factor (last column), the highest average BLI data point (i.e., 20.30) was selected and divided by the average BLI data at each concentration. As a result, samples with 2E11 vp / ml, 8E10 vp / ml, and 3E10 vp / ml generated normalization factors of 1.00, 1.53, and 3.52, respectively.

[0196] Table 3.

[0197]

[0198] 2E11 vp / ml, 8E10 vp / ml, and 3E11 vp / ml of captured AAV capsids were lysed and produced the following signal curves in a ssDNA assay according to Example 7A, except that the wavelength was read at 60 seconds. Fig.17 The results are shown in .

[0199] By using Gator software, we extracted Fig.16 The data points of each signal curve in the , and the repeated BLI data are averaged. The average BLI data is then subtracted from the PBS control to generate the net BLI signal. The net BLI signal is multiplied by the normalization factor from Table 3 to generate the normalized offset value in Table 4.

[0200] Table 4.

[0201]

[0202] The normalized offset values ​​were then used to determine the empty / full ratios of the samples at different concentrations. Fig.18 ), the determined sample empty / intact ratios at 2E11 vg / ml, 8E10 vg / ml and 3E10 vg / ml were 54.7%, 59.6% and 59.9%, respectively. Overall, the average empty / intact ratio was 58.1% and the CV was only 5.0%.

[0203] In this example, samples containing AAV capsids of the same empty / complete ratio but at different concentrations were prepared and tested to see if normalization of BLI data could be reliably used to determine the empty / complete ratio of a sample. In summary, by using the normalization factor, the empty / complete ratios of each sample determined at different AAV concentrations were very similar to each other (CV of 5.0%). The results show that the normalization procedure can be used to compensate for sample AAV concentration differences and reliably calculate the empty / complete ratio of AAV capsids.

[0204] Example 13. Preparation of anti-DNA-HRP conjugate

[0205] Anti-ssDNA / dsDNA IgG (Absolute Antibody, M3D8, mouse IgG, catalog number Ab00347-1.1) (1 mg in 1 mL PBS) was reacted with SMCC (molar coupling ratio MCR=8, 20 mg / mL in DMF, 0.89 μL, ThermoScientific) for 1 hour and then stained with Zeba TM Desalting spin column (7k MWCO, Thermo Scientific) purification. Horseradish peroxidase (HRP, Thermo Scientific, MCR = 6 against -DNA) (1.6 mg, in 80 μL PBS) was reacted with Traut reagent (MCR = 8, 20 mg / mL in water, 1.65 μL, Thermo Scientific) for 1 hour, and then Zeba TM Desalting centrifugal column purification. The resulting anti-DNA-SMCC and HRP-thiol solutions were mixed and reacted for 3 hours, and then quenched with 12.5 μL of N-ethylmaleimide (NEM, Thermo Scientific, catalog number 23030) in water (20 mg / mL) for 0.5 hours. The conjugate was purified by Zeba TM After desalting the spin column, anti-DNA-HRP in PBS was obtained for ssDNA detection solution.

[0206] Example 14: Preparation of streptavidin-coated magnetic particles for AAV capture

[0207] Magnetic particles are used as a platform for streptavidin conjugation. To this end, 1 mg of acid-coated magnetic particles (OceanNanoTech, 1 μm in diameter) are suspended in 1 mL of 50 mM MES buffer (pH=6) containing 4 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mM N-hydroxysuccinimide (NHS), and reacted for 30 minutes. The magnetic particles are then separated using a magnet and the supernatant is discarded. Next, 50 μg of streptavidin (SA, Agilent) in PBS buffer (pH=7.4, 1 mL) is added to the magnetic particles and reacted for 2 hours. The magnetic particles are separated again using a magnet and the supernatant is discarded. In order to quench excess NHS esters, 50 mM Tris buffer (pH=8, 1 mL) is added to the magnetic particles and reacted for 30 minutes. Finally, the magnetic particles are separated using a magnet and the supernatant is discarded. A suspension of magnetic particles conjugated with 50 μg / mL SA was obtained by adding 1 mL of PBS buffer.

[0208] Example 15. Preparation of anti-AAV magnetic particles

[0209] A 7 μL aliquot of 0.1 mg / ml biotin-anti-AAVX (Thermo, Capture Select) was added to 100 μL streptavidin-coated magnetic particles (Example 14) at 1 mg / ml and incubated for 30 minutes at room temperature with orbital mixing. The particles were then washed by precipitation with a magnet, removal of the supernatant, and resuspension with 200 μL PBS. Three wash cycles were performed.

[0210] Example 1 6. Measuring DNA content in AAV samples

[0211] Magnetic particle capture

[0212] Two AAV samples (Virovek, complete #AAV8-CMV-GFP) were titrated initially with GatorBio AAV probe (#160017) according to the manufacturer's protocol and diluted to 1.4 × 10 11 vp / ml and 6.1×10 9 vp / ml. A 25 μL aliquot of anti-AAV coated magnetic particles (Example 15) was added to 500 μL AAV sample and incubated at room temperature (RT) for 30 minutes. The particles were then precipitated magnetically. 100 μL of the supernatant was removed and assayed with the GatorBio AAV probe to determine the uncaptured capsids in the supernatant. The results from the starting capsid concentrations before and after magnetic particle capture enabled the calculation of the captured capsids, which were 3.7×10 10 vp and 3.1×109 The remaining supernatant was then removed from the sample.

[0213] Lysis

[0214] 100 μL of lysis reagent (PBS + 3% Tween 20) was added to the magnetic particles and heated at 95 μ for 5 minutes.

[0215] ssDNA assay

[0216] After lysis, 100 μL of the lysate containing the lysed AAV was transferred to a microplate and ssDNA was measured using the ssDNA assay in the GatorBio AAV Ratio Kit (#35004) using a Gatro Plus BLI reader following the manufacturer's protocol. Fig.19 The results are shown in .

[0217] This example illustrates that the combination of magnetic particle capture, lysis, and ssDNA detection with a BLI biosensor can be used to measure DNA in AAV samples.

[0218] Example 17: Preparation of Magnetic Particles Coated with Biotin-Anti-AAVX

[0219] Magnetic beads from different suppliers with streptavidin surface coating were used. The amount of beads used was estimated from the binding capacity reported by the supplier. For Dynabeads MyOne magnetic beads with streptavidin coating, the particles contained 10 mg / mL (approximately 7-10 × 10 9 Beads / ml). Resuspend the beads in the vial by vortexing for 10 to 20 seconds. Pipette 10 μL of the pellet into a 96-well plate or tube. Mix an equal volume of wash buffer (PBS) with the beads and remove the supernatant using magnetic separation. Repeat the wash step 3 times. Typically, 0.1 mg of Dynabeads (10 μL) is able to bind 2 μg of biotinylated protein. Add an estimated amount of biotin anti-AAVx conjugate (CaptureSelect TM ) was added to the suspended streptavidin surface functionalized magnetic particles and incubated at room temperature with gentle mixing for 15 minutes. The mixture was placed in a magnetic separation device to remove the supernatant. The coated beads were washed 3 to 4 times with wash buffer and resuspended in 200 μL of PBS for AAV capsid capture experiments.

[0220] Example 18. Preparation of magnetic particles coated with biotin-single-stranded DNA binding protein (SSB)

[0221] Similar to Example 13, Dynabeads MyOne magnetic beads with streptavidin coating were washed and prepared according to the above steps. The washed beads were treated with biotinylated SSB, and the estimated value was 0.1 mg of Dynabeads (10 μL) bound to 2 μg of biotinylated protein. The mixture was incubated at room temperature for 15 minutes by gentle mixing. Finally, the supernatant was removed by magnetic separation, and the coated beads were washed 3 to 4 times with washing buffer and resuspended in 200 μL PBS for ssDNA binding assay.

[0222] Example 19. Detection of ssDNA using magnetic particles

[0223] AAV capture procedure:

[0224] Calculate the volume of empty and complete AAV capsids required to prepare 200 μL of 2E11 vp / ml at 0%, 20%, 40%, 60%, 80% and 100% complete capsid ratios. Dilutions of the capsids can be made using PBS.

[0225] - Test AAV samples (unknown E / F ratio) were adjusted to 2E11 vp / ml and 200 μL was used for the assay.

[0226] • Optional: As a positive control, prepare 200 μL of 10 ng / ml M13 ssDNA in PBS, and use PBS only as a reference control.

[0227] • Transfer 200 μL of each sample into an Eppendorff tube.

[0228] · Place 10 μL of CaptureSelect TM Magnetic particles were added to each Eppendorff tube.

[0229] - Vortex the tube briefly and incubate the sample at room temperature for 30 minutes.

[0230] After 30 minutes, place the tubes in the magnetic rack and wait 30 seconds.

[0231] • Pipetting was performed to remove as much sample solution as possible by using magnetic separation.

[0232] • Add 500 μL of PBS to the tube and repeat the wash step 3 times.

[0233] - After washing, add 200 μL of lysis buffer (PBS containing 3% Tween 20) to each tube.

[0234] AAV lysis procedure :

[0235] • Place the tube into a heat block that has been set to 70 °C and wait 5 minutes to release the ssDNA.

[0236] After 5 minutes, set the tubes aside and allow them to cool at room temperature.

[0237] Place the Eppendorff tube in the magnetic rack and wait 30 seconds.

[0238] • Pipet to remove the solution from the beads by magnetic separation and transfer the solution alone to a new Eppendorff tube.

[0239] ssDNA detection procedure :

[0240] • Add 10 μL of SSB-coated magnetic particles to each tube.

[0241] - Vortex the tube briefly and incubate the sample at room temperature for 30 minutes.

[0242] After 30 minutes, place the tubes in the magnetic rack and wait 30 seconds.

[0243] • Pipetting was performed to remove as much sample solution as possible by using magnetic separation.

[0244] • Add 500 μL of PBS to the tube and repeat the wash step 3 times.

[0245] • After washing, add 200 μL of PBS to the tube.

[0246] • Add 10 μL of anti-ssDNA / dsDNA-HRP conjugate to each tube.

[0247] Vortex briefly and incubate at room temperature for 15 minutes.

[0248] After 15 minutes, place the tubes in a magnetic rack for 30 seconds.

[0249] • Pipetting was performed to remove the solution and retain the beads by using magnetic separation.

[0250] - Add 500 μL of PBS to the tube containing the beads and repeat the wash step 3 times.

[0251] - After washing, add 100 μL of PBS to the tube.

[0252] Vortex briefly and transfer samples to desired wells of a 96-well flat-bottom polystyrene microplate.

[0253] • Add 100 μL of TMB substrate to each well.

[0254] Tap or shake the plate to ensure complete mixing.

[0255] Add 50 μL of stop solution and terminate the reaction after 15 minutes.

[0256] • Measure OD at 450 nm.

[0257] • Plot the OD readings at 450 nm versus % capsid to generate a standard curve. The OD readings at 450 nm of the test samples can be used to determine % capsid from the standard curve.

[0258] Example 20. Preparation of anti-ssDNA / dsDNA antibodies conjugated with FITC

[0259] The anti-ssDNA / dsDNA antibody was conjugated to FITC and purified using the EZLabel™ Protein FITC Labeling Kit from BioVision.

[0260] Reconstitute one vial of EZLabel with 5 μL to 10 μL of DMSO TM FITC.

[0261] · Refactored EZLabel TM The FITC solution was added to an Eppendorff tube containing 100 μL (up to 1 mg) of anti-ssDNA IgG Ab and vortexed briefly.

[0262] - Incubate for 1 hour at room temperature (RT) on a rotary shaker.

[0263] After incubation, add 20 μL EZLabel TM Quench buffer to quench the reaction.

[0264] - Incubate at room temperature for a further 30 minutes.

[0265] Prepare and wash EZLabel™ spin columns according to the manufacturer's instructions.

[0266] Load the FITC-labeled mixture onto the EZLabel™ spin column.

[0267] • Spin the column and elute the purified FITC-labeled anti-ssDNA / dsDNA antibody.

[0268] Example 21. ssDNA detection by using fluorescent labels

[0269] • Add 10 μL of SSB-coated magnetic particles to each tube.

[0270] - Vortex the tube briefly and incubate the sample at room temperature for 30 minutes.

[0271] After 30 minutes, place the tubes in the magnetic rack and wait 30 seconds.

[0272] • Pipetting was performed to remove as much sample solution as possible by using magnetic separation.

[0273] • Add 500 μL of PBS to the tube and repeat the wash step 3 times.

[0274] • After washing, add 200 μL of PBS to the tube.

[0275] Add 10 μL of FITC-labeled anti-ssDNA / dsDNA antibody to each tube.

[0276] Vortex briefly and incubate at room temperature for 15 minutes.

[0277] After 15 minutes, place the tubes in a magnetic rack for 30 seconds.

[0278] • Pipetting was performed to remove the solution and retain the beads by using magnetic separation.

[0279] - Add 500 μL of PBS to the tube containing the beads and repeat the wash step 3 times.

[0280] - After washing, add 100 μL of PBS to the tube.

[0281] Vortex briefly and transfer samples to desired wells of a white 96-well flat bottom white opaque microplate.

[0282] Tap or shake the plate to ensure complete mixing.

[0283] ·Measure the fluorescence of the wells with excitation and emission wavelengths of 494 nm and 520 nm respectively.

[0284] • Plot the fluorescence readings versus % capsid to generate a standard curve. The fluorescence readings of the test samples can be used to determine % capsid from the standard curve.

[0285] The invention and the manner and process of making and using the same are now described in full, clear, concise and precise terms to enable any person skilled in the art to make and use the invention. It should be understood that the foregoing describes preferred embodiments of the invention and that modifications may be made therein without departing from the scope of the invention as set forth in the claims. In order to particularly point out and distinctly claim the subject matter regarded as inventive, the specification ends with the following claims.

Claims

1. A method for determining the DNA concentration in a DNA virus, the method comprising the following steps: (a) obtaining a first probe having an anti-viral antibody immobilized on the tip of the probe, wherein the virus is adeno-associated virus (AAV), adenovirus or herpes simplex virus type 1 (HSV-1); (b) dipping the first probe tip in a sample solution containing a virus sample to capture the virus on the probe under defined binding conditions; (c) immersing the first probe in a washing solution to wash the tip of the first probe; (d) immersing the first probe in a lysis solution, cleaving the virus from the tip of the first probe to obtain single-stranded DNA (ssDNA), and then removing the first probe from the lysis solution; (e) immersing a second probe comprising a first protein on the tip of the probe in the lysis solution, wherein the first protein binds to ssDNA; (f) immersing the second probe in a reagent solution containing a second protein labeled with HRP, wherein the second protein binds to ssDNA; (g) immersing the second probe in an HRP substrate solution to allow the substrate to bind to the HRP bound to the second probe for a period of time; as well as (h) determining the ssDNA concentration in the sample by measuring the wavelength shift due to light interference, and quantifying the wavelength shift against a calibration curve to determine the ssDNA concentration.

2. A method for determining the DNA concentration in a DNA virus, the method comprising the following steps: (a) obtaining a first probe having a fixed amount of an anti-viral antibody immobilized on the tip of the probe, wherein the virus is AAV, adenovirus or HSV-1; (b) immersing the first probe tip in a sample solution containing a virus sample to capture the virus on the probe; (c) immersing the first probe in a washing solution to wash the tip of the first probe; (d) immersing the first probe in a lysis solution, cleaving the virus from the tip of the first probe to obtain single-stranded DNA (ssDNA), and then removing the first probe from the lysis solution; (e) immersing a second probe comprising a first protein on the tip of the probe in the lysis solution, wherein the first protein binds to ssDNA; (f) immersing the second probe in a reagent solution containing a biotin-labeled second protein, wherein the second protein binds to ssDNA; (g) immersing the second probe in a conjugate solution containing a conjugate containing streptavidin and horseradish peroxidase (HRP) to allow the conjugate to bind to the second probe, (h) immersing the second probe in an HRP substrate solution to allow the substrate to bind to the HRP in the conjugate for a period of time; as well as (i) determining the ssDNA concentration in the sample by measuring the wavelength shift due to light interference, and quantifying the wavelength shift against a calibration curve to determine the ssDNA concentration.

3. The method of claim 1 or 2, wherein the virus is AAV.

4. The method according to claim 1 or 2, wherein in step (b), the virus is captured until the maximum binding capacity of the antiviral antibody on the probe is reached.

5. The method according to claim 1 or 2, wherein the first protein and the second protein are independently antibodies against ssDNA or single-stranded DNA binding proteins (SSBs). The method according to claim 1 or 2, wherein the diameter of the tip surface of the second probe is ≤ 5 mm.

7. The method of claim 2, wherein the conjugate in step (g) comprises: (i) streptavidin, HRP and a polymer, or (ii) streptavidin and cross-linked HRP, or (iii) cross-linked streptavidin and HRP.

8. The method of claim 7, wherein the conjugate comprises streptavidin, HRP, and a polymer, and the polymer has a molecular weight of more than 1 million.

9. The method of claim 8, wherein the polymer is a cross-linked copolymer of sucrose and epichlorohydrin.

10. A method for measuring the percentage of intact viral capsids in a viral sample; the method comprising first determining the ssDNA concentration in AAV according to the method of claim 1 or 2, and then converting the ssDNA concentration to % of intact AAV using a previously established calibration curve with DNA concentrations plotted against standards of % of intact viral capsids.

11. A method for measuring the percentage of intact viral capsids in a sample, the method comprising the steps of: (a) obtaining a first probe having a fixed amount of an anti-DNA virus antibody fixed on the tip of the probe; (b) immersing the first probe tip in a sample solution containing a DNA virus sample, to capture the virus capsid on the probe for a period of time under defined binding conditions, thereby measuring a first wavelength shift caused by light interference due to the bound virus on the probe; (c) determining a normalization factor based on a comparison of the first wavelength shift of the sample with (i) the first wavelength shift of the calibrants or (ii) an average of the first wavelength shifts of all calibrants; (d) immersing the first probe in a washing solution to wash the tip of the first probe; (e) immersing the first probe in a cleavage solution, cleaving the AAV from the tip of the first probe to obtain single-stranded DNA (ssDNA), and then removing the first probe from the cleavage solution; (f) immersing a second probe comprising a first protein on the tip of the probe in the lysis solution, wherein the first protein binds to ssDNA; (g) immersing the second probe in a reagent solution containing a second protein labeled with HRP, wherein the second protein binds to ssDNA; (h) immersing the second probe in an HRP substrate solution to allow the substrate to bind to the HRP bound to the second probe for a period of time, and measuring the second wavelength shift caused by light interference; (i) applying the normalization factor to the second wavelength shift to produce a normalized second wavelength shift; as well as (i) quantifying the normalized second wavelength shift against a calibration curve with normalized wavelength shifts plotted against % of intact AAV capsids to determine the percentage of intact AAV capsids in the sample.

12. The method of claim 11, wherein the normalization factor in step (c) is determined by comparing the first wavelength shift of the sample with the average of the first wavelength shifts of all calibrants.

13. The method of claim 12, wherein the calibration curve is established by measuring the normalized shift of calibrator samples having a known percentage of intact AAV capsids and plotting the normalized wavelength shift against the percentage of intact AAV capsids.

14. The method according to any one of claims 11 to 13, wherein the virus is AAV.

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