阅读说明：本技术 Aav蛋白外壳的检测方法 (Detection method of AAV protein coat ) 是由 王慧 蒋威 郑静 肖啸 吴侠 杜增民 王利群 赵阳 陈晨 郑浩 于 2021-07-30 设计创作，主要内容包括：本公开涉及AAV蛋白外壳的检测方法以及可应用于该检测方法的试剂盒。本公开的检测方法可以准确定量AAV蛋白外壳的浓度,具有高灵敏度。(The present disclosure relates to methods for detection of AAV protein coat and kits applicable thereto. The detection method disclosed by the invention can accurately quantify the concentration of the AAV protein coat and has high sensitivity.)

A method for detecting an AAV protein coat, comprising:

1) linking the antibody with a solid phase carrier to form a solid phase antibody;

2) adding an antigen, and combining the antigen and the solid-phase antibody to form a solid-phase antigen-antibody complex;

3) adding a biotinylated antibody, and combining the biotinylated antibody and the antigen on the solid-phase antigen-antibody complex to form a sandwich;

4) adding a labeled probe bound to an enzyme, and allowing the labeled probe to bind to a biotinylated antibody;

5) adding a substrate for color development, wherein the substrate is subjected to color change under the catalysis of enzyme;

6) and determining the concentration of the antigen to be detected according to the color change of the substrate.

2. The assay of claim 1, wherein the amino acid sequence of the AAV protein coat differs from the amino acid sequence of SEQ ID NO: 9 has at least 80%, 85%, 90%, 95% identity; preferably, the AAV protein coat has an amino acid sequence as set forth in SEQ ID NO: shown at 9.

3. The detection method according to claim 1 or 2, wherein the antibody has a K of less than 1.0E-12M_DBinds to the AAV protein capsid.

4. The assay of any one of claims 1 to 3 wherein the antibody comprises a VH CDR1-3 of the heavy chain variable region and a VL CDR1-3 of the light chain variable region, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 being identical to SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, and the amino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 are at least 80%, 85%, 90%, 95% identical to SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6 are at least 80%, 85%, 90%, 95% identical.

5. The assay of claim 4, wherein the amino acid sequences of the VH CDR1, VH CDR2, and VH CDR3 are set forth in SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 are respectively shown in SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: and 6.

6. The detection method according to any one of claims 1 to 3, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being identical to the amino acid sequence of SEQ ID NO: 7, and the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 95% identity with SEQ ID NO: 8, preferably, the amino acid sequence of the AAV protein capsid is at least 80%, 85%, 90%, 95% identical to SEQ ID NO: 9 has at least 80%, 85%, 90%, 95% identity.

7. The assay of claim 6, wherein the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 7, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8.

8. The detection method according to any one of claims 1 to 7, wherein the antibody is a chimeric antibody, a humanized antibody or a fully human antibody.

9. The detection method according to any one of claims 1 to 8, wherein the detection method is a qualitative detection method or a quantitative detection method, preferably a quantitative detection method.

10. The detection method according to any one of claims 1 to 9, wherein the labeled probe is a biotin or avidin labeled probe.

11. The detection method according to any one of claims 1 to 10, wherein the enzyme is horseradish peroxidase.

12. The detection method according to any one of claims 1 to 11, wherein the chromogenic substrate is 3,3',5,5' -Tetramethylbenzidine (TMB) or Diaminobenzidine (DAB).

13. The detection method according to any one of claims 1 to 12, wherein the solid phase carrier is selected from the group consisting of: polyvinyl chloride, polystyrene, nylon, polypropylene, polyacrylamide, and cellulose.

14. A kit, comprising:

an antibody comprising a VH CDR1-3 of a heavy chain variable region and a VL CDR1-3 of a light chain variable region, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 are set forth in SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 are respectively shown in SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification;

the antibody being biotinylated;

optionally a label probe bound to an enzyme; and the number of the first and second groups,

optionally a chromogenic substrate capable of reacting with the enzyme.

15. The kit of claim 14, wherein the labeled probe is a biotin or avidin labeled probe.

16. The kit of claim 14 or 15, wherein the enzyme is horseradish peroxidase.

17. The kit according to any one of claims 14 to 16, wherein the chromogenic substrate is 3,3',5,5' -Tetramethylbenzidine (TMB) or Diaminobenzidine (DAB).

Technical Field

The present disclosure relates to methods for detection of AAV protein coat and kits applicable thereto.

Background

Adeno-associated viruses (AAV) are a class of tiny, non-enveloped and icosahedral-structured viruses. The viral particles have a diameter of between 20 and 26nm and contain a linear single-stranded DNA genome of about 4.7kb in size. AAV was originally named because it is a contaminant found in purified adenovirus fluid. Most adults have been infected with AAV virus, but the virus has not been found to be a causative agent of any disease.

Recombinant adeno-associated virus (rAAV) is a novel gene vector which is further modified on the basis of non-pathogenic wild AAV, is regarded as one of the most promising gene transfer vectors due to the characteristics of good safety, wide host cell range, low immunogenicity, long time for expressing foreign genes in vivo and the like, and is widely applied to gene therapy research in the world. A feature of some of the current new AAV serotypes is that transduction efficiency of the heart, muscle and CNS is greatly improved by systemic gene delivery. Others are effective in liver gene transfer in small animals.

These findings provide insights into gene therapy for a number of genetic and metabolic diseases. In addition to identifying new serotypes, genetic modifications can further improve the clinical utility of AAV vectors. One problem with the use of AAV vectors is their extensive tissue tropism, which often leads to gene transfer in unwanted tissues and potential safety hazards for ectopic gene expression. In order to improve the tissue specificity and tissue targeting ability of AAV viral particles, random mutagenesis of the capsid gene is one of the most effective methods to enhance capsid diversity and alter capsid tropism to target tissue, in addition to rational engineering. Directed evolution (e.g., DNA shuffling) is one of the most recently developed methods of genetic engineering of AAV capsid genes.

International patent application WO 2019241324 a1 relates to a synthetic adeno-associated viral protein capsid targeted to the liver obtained by directed evolution, named AAVXL 32.1. However, there is no monoclonal antibody and detection method that specifically recognizes the protein capsid (AAVXL32.1), and accurate quantitative detection thereof is impossible.

Disclosure of Invention

To address the above-described deficiencies in the prior art, in a first aspect, the present disclosure provides a method of detecting an AAV protein coat, comprising: 1) linking the antibody with a solid phase carrier to form a solid phase antibody; 2) adding an antigen, and combining the antigen and the solid-phase antibody to form a solid-phase antigen-antibody complex; 3) adding a biotinylated antibody, and combining the biotinylated antibody and the antigen on the solid-phase antigen-antibody complex to form a sandwich; 4) adding a labeled probe bound to an enzyme, and allowing the labeled probe to bind to a biotinylated antibody; 5) adding a substrate for color development, wherein the substrate is subjected to color change under the catalysis of enzyme; 6) and determining the concentration of the antigen to be detected according to the color change of the substrate.

The detection method disclosed by the invention can accurately quantify the concentration of the AAV protein coat and has high sensitivity.

In one embodiment, the amino acid sequence of the AAV protein coat is identical to SEQ ID NO: 9 has at least 80%, 85%, 90%, 95% identity. In a preferred embodiment, the amino acid sequence of the AAV protein capsid is as set forth in SEQ ID NO: shown at 9.

In one embodiment, the antibody is raised to a K of less than 1.0E-12M_DBinds to the AAV protein capsid.

In one embodiment, the antibody comprises VH CDR1-3 of the heavy chain variable region and VL CDR1-3 of the light chain variable region, the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3, respectively, and SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, and the amino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 are at least 80%, 85%, 90%, 95% identical to SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6 are at least 80%, 85%, 90%, 95% identical.

In one embodiment, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 are set forth in SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 are respectively shown in SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: and 6.

In one embodiment, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is identical to the amino acid sequence of SEQ ID NO: 7, and the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 95% identity with SEQ ID NO: 8 have at least 80%, 85%, 90%, 95% identity.

In one embodiment, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 7, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8.

In one embodiment, the antibody is a chimeric antibody, a humanized antibody or a fully human antibody.

In one embodiment, the detection method is a qualitative detection method or a quantitative detection method, preferably a quantitative detection method.

In one embodiment, the labeled probe is a biotin or avidin labeled probe.

In one embodiment, the enzyme is horseradish peroxidase.

In one embodiment, the chromogenic substrate is 3,3',5,5' -Tetramethylbenzidine (TMB) or Diaminobenzidine (DAB).

In one embodiment, the solid support is selected from the group consisting of: polyvinyl chloride, polystyrene, nylon, polypropylene, polyacrylamide, and cellulose.

In a second aspect, the present disclosure provides a kit comprising: an antibody comprising a VH CDR1-3 of a heavy chain variable region and a VL CDR1-3 of a light chain variable region, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 are set forth in SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 are respectively shown in SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification; the antibody being biotinylated; optionally a label probe bound to an enzyme; and, optionally, a chromogenic substrate capable of reacting with the enzyme.

In one embodiment, the labeled probe is a biotin or avidin labeled probe.

In one embodiment, the enzyme is horseradish peroxidase.

In one embodiment, the chromogenic substrate is 3,3',5,5' -Tetramethylbenzidine (TMB) or Diaminobenzidine (DAB).

Drawings

Figure 1 shows the SDS-PAGE assay for mAb002 antibody.

Figure 2 shows SEC-HPLC determination of mAb002 antibody.

Fig. 3 shows the amino acid sequences of the heavy chain variable region and the light chain variable region of mAb002 antibody.

FIG. 4 shows a sandwich ELISA schematic.

Figure 5 shows a standard curve for a sandwich ELISA assay.

Fig. 6 shows the results of the detection of kinetic parameters of mAb002 antibody.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") in this context.

As used herein, the term "antibody" refers to an intact antibody or an antibody fragment that competes with the intact antibody for binding to an antigen. Antibody fragments include, but are not limited to: fab, Fab ', F (ab') 2, Fv, scFv, Fd, diabodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. In some embodiments, the antibody fragment is produced by enzymatic reaction or chemical cleavage of an intact antibody. In some embodiments, the antibody fragment is produced by recombinant DNA techniques.

Herein, the term "monoclonal antibody" refers to a homogeneous immunoglobulin molecule, abbreviated as monoclonal antibody, secreted by a single clonal lymphocyte against a particular antigenic determinant on an antigenic molecule. In some embodiments, the monoclonal antibody is secreted by the hybridoma. In some embodiments, the hybridomas are produced according to methods known to those of skill in the art, e.g., Kohler and Milstein (1975) Nature,256: 495-499. In some embodiments, monoclonal antibodies are produced using recombinant DNA methods (as described in U.S. patent No. 4,816,567).

As used herein, the term "chimeric antibody" refers to an antibody that is composed of components from at least two different sources. In some embodiments, a chimeric antibody comprises an antibody moiety from a first species fused to another molecule, e.g., an antibody moiety from a second species. In some embodiments, the chimeric antibody comprises an antibody portion from a non-human animal fused to an antibody portion from a human. In some embodiments, the chimeric antibody comprises all or part of a variable region of an antibody from a non-human animal fused to a constant region of an antibody from a human.

As used herein, the term "humanized antibody" refers to an antibody produced by the modification and re-expression of a non-human (e.g., murine) monoclonal antibody using gene cloning and DNA recombination techniques. Most of the amino acid sequences of the humanized antibody are replaced by human sequences, so that the affinity and the specificity of the parent non-human (such as mouse) monoclonal antibody are basically reserved, the heterogeneity is reduced, and the humanized antibody is favorably applied to a human body. Humanized antibodies are generally less immunogenic to humans than non-humanized antibodies.

Herein, the term "fully human antibody" refers to an immunoglobulin comprising both human framework regions and human CDRs. The constant regions need not be present intact, but if present they are substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical.

An antibody "specifically binds" to an antigen when it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, the antibody comprises an antigen binding site that specifically binds to a particular epitope. In some embodiments, an antibody is considered to specifically bind to an antigen when the dissociation constant (KD) is less than 1 μ Μ, less than 100nM, less than 10nM, or less than 1 nM.

In some embodiments, the antibody is modified to alter one or more of its properties. In some embodiments, the modified antibody may have advantages, such as increased stability, relative to an unmodified antibody. In some embodiments, the antibody is modified by altering the glycosylation state of the antibody, such as by altering the number, type, linkage, and/or position of sugar chains on the antibody.

As used herein, the term "percent identity" or "% identity" refers to the percentage of amino acids that are identical between at least two polypeptide sequences aligned using the Basic Local Alignment Search Tool (BLAST) engine.

Antibodies and methods for making antibodies are well known in the art.

In some embodiments, monoclonal antibodies are produced by standard techniques. In some embodiments, the monoclonal antibody is produced by a hybridoma-based method. In some embodiments, a suitable animal, such as a mouse, rat, hamster, monkey, or other mammal, is immunized with an immunogen to produce antibody-secreting cells. In some embodiments, the antibody-secreting cell is a B cell, such as a lymphocyte or a spleen cell. In some embodiments, lymphocytes (e.g., human lymphocytes) are immunized in vitro to produce antibody-secreting cells.

In some embodiments, antibody-secreting cells are fused to "immortalized" cell lines, such as myeloid-type cell lines, to produce hybridoma cells. In some embodiments, successfully fused hybridoma cells are selected using HAT selective medium containing three components, hypoxanthine (H), aminopterin (a), and thymidine (T). In some embodiments, hybridoma cells that secrete the desired antibody are screened, e.g., by ELISA, and such cells are then subcloned and cultured using standard methods. In some embodiments, the desired antibody is an antibody specific for an antigen of interest. In some embodiments, such cells may also be cultured in a suitable animal host in vivo as an ascites tumor. In some embodiments, the monoclonal antibody is isolated from hybridoma culture medium, serum, or ascites fluid using standard isolation procedures (e.g., affinity chromatography).

In some embodiments, the human monoclonal antibodies are generated using a display-based method. In some embodiments, the human monoclonal antibodies are generated using phage display technology. Some exemplary Antibody Phage Display Methods are known to those of skill in the art, such as Hoogenboom, Overview of Antibody phase-Display Technology and Its Applications, Methods in Molecular Biology: Antibody phase-Display: Methods and Protocols, (2002)178:1-37 (O' Brien and Aitken, Human Press, Totowa, NJ).

In some embodiments, the affinity of an antibody for a particular antigen is increased by affinity maturation (or "directed evolution") of the antibody in vitro.

In some embodiments, the monoclonal antibody is produced by recombinant techniques, as described in U.S. Pat. No. 4,816,567. In some embodiments, nucleic acids encoding monoclonal antibody chains are cloned and expressed in a suitable host cell. In some embodiments, RNA is prepared from cells expressing the desired antibody (e.g., mature B cells or hybridoma cells) using standard methods, and then cDNA is prepared from the RNA using standard methods. In some embodiments, cDNA encoding a heavy or light chain polypeptide is amplified by PCR using oligonucleotide primers. In some embodiments, the cDNA is cloned into a suitable expression vector, which is then transformed or transfected into a suitable host cell. Some exemplary host cells include: escherichia coli, COS cells, Chinese Hamster Ovary (CHO) cells, and myeloma cells.

In some embodiments, the ability of an antibody to bind to an antigen (e.g., an AAV protein capsid) is determined using conventional methods for detecting binding of an antibody to an antigen.

For example, in some embodiments, the ability of a monoclonal antibody to bind to an AAV protein capsid is determined by standard immunoblotting methods, such as western blotting. In some embodiments, the ability of a monoclonal antibody to bind to an AAV protein capsid is determined using a competitive binding assay. In some embodiments, the competitive binding assay is performed using ELISA. In some embodiments, the binding kinetics (e.g., rate constant) or binding affinity (e.g., association or dissociation constant) of an antibody is determined using a binding assay.

The monoclonal antibodies of the present disclosure may be subject to "conservative substitutions" of amino acids. "conservative substitution" of an amino acid refers to the replacement of an amino acid in a polypeptide with another amino acid having similar properties (e.g., size or charge). Conservative substitutions of amino acids are known in the art. In some embodiments, a polypeptide comprising an amino acid conservative substitution retains at least one activity of the unsubstituted polypeptide. In one embodiment, the monoclonal antibodies of the disclosure may have conservative substitutions of amino acids within the same group as: a) glycine and alanine; b) valine, isoleucine, leucine and proline; c) aspartic acid and glutamic acid; d) asparagine and glutamine; e) serine, threonine lysine, arginine, and histidine; f) phenylalanine, tryptophan, and tyrosine; g) methionine and cysteine.

In one embodiment, AAV protein capsids are qualitatively or quantitatively detected using a monoclonal antibody of the disclosure. In one embodiment, AAV protein capsids are qualitatively or quantitatively detected using a double antibody sandwich ELISA using monoclonal antibodies of the disclosure. In one embodiment, the method for detecting the antigen concentration by using the double antibody sandwich ELISA method comprises the following steps: linking the antibody with a solid phase carrier to form a solid phase antibody; adding an antigen, and combining the antigen and the solid-phase antibody to form a solid-phase antigen-antibody complex; adding a biotinylated antibody, and combining the biotinylated antibody and the antigen on the solid-phase antigen-antibody complex to form a sandwich; adding horseradish peroxidase labeled streptavidin, wherein the horseradish peroxidase labeled streptavidin is combined with a biotinylated antibody; adding a substrate for color development, wherein the substrate is subjected to color change under the catalysis of horseradish peroxidase; and determining the concentration of the antigen to be detected according to the color change of the substrate.

The present disclosure is described in further detail below with reference to the accompanying drawings and examples. The following examples are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure. The experimental procedures, in which the specific conditions are not indicated in the examples, are carried out according to the conventional conditions known in the art or according to the conditions recommended by the manufacturer.

Examples

Example 1: production of antigens

HEK293 cells were transfected with both plasmids to produce the complete hollow AAV protein capsid (AAVXL32.1) as antigen. AAV rep/cap plasmid was used: phepper: polyethyleneimine (PEI) ═ 1:1:2, mixed in Opti-MEM, left to stand for 12min, and the transfection solution was added to the suspension HEK293 cells. After 24h of carbon dioxide incubator, the old medium was discarded and new medium was added. After further culturing for 24h, the supernatant and cells were collected. 2mM MgCl was added₂And carrying out ultrasonic disruption. After disruption at 300W for 4min, the cells were disrupted. After adding 50U/mL of Benzonase enzyme and incubating at 37 ℃ for 1h, cell debris was removed by centrifugation at 8000 Xg for 15 min. The supernatant was filtered sequentially using 0.8 μm and 0.45 μm filters.

The Heparin-Sepharose HP (Heparin-Sepharose HP) was used for capture, the Heparin column was equilibrated with an equilibration solution (20mM Tris, 20mM NaCl, Pluronic F-68 (0.001% (w/v), pH7.5), the filtered supernatant was sample bound after equilibration, the equilibration solution was used after binding was completed, the eluent (20mM Tris, 1M NaCl, Pluronic F-68 (0.001% (w/v), pH7.5) was used for elution, the eluent was concentrated using an ultrafiltration tube (100KD) for exchange, and the final solution for exchange was PBS + Pluronic F-68 (0.001% (w/v) to obtain an antigen (AAVXL32.1 protein capsid), the concentration of the antigen was measured by Nanoderop (Thermofisher, Nanoderop 2000C Spectroscopy) at A280nm and was 1 mg/ml.

Example 2: obtaining hybridoma cells

Immunizing animals

6-8 week-old female Balb/c mice were immunized with the antigen obtained in example 1 to generate sensitized B lymphocytes. Mice were sacrificed by enucleation and exsanguination, spleens were aseptically removed, and splenocytes suspensions were prepared by crushing and grinding in dishes.

Production of hybridoma cells

Syngeneic myeloma cells and mouse splenocytes were mixed as 1:1, and adding a fluxing agent polyethylene glycol. Under the action of polyethylene glycol, the B lymphocyte and myeloma cell are fused to form hybridoma cell. Following fusion of B lymphocytes and myeloma cells, five cell types are generated: unfused splenocytes, unfused myeloma cells, splenocyte-splenocyte fusions, myeloma cell-myeloma cell fusions, splenocyte-myeloma cell hybrids (hybridoma cells). Then, using hybridoma cell screening technology, hybridoma cells were isolated using HAT selective medium (purchased from Solarbio, cat # H0262) containing hypoxanthine (H), aminopterin (a) and thymidine (T).

Screening of hybridoma Positive clones

The culture medium containing the hybridoma cells is diluted into multi-well plates such that each well contains only one hybridoma cell. Screening out positive hybridoma cell capable of producing the required monoclonal antibody by ELISA method, and cloning and amplifying. Hybridoma cells were cultured in 1mL of a medium in a 24-well plate using 10% fetal bovine serum and 1 xhat added to DMEM as a basic culture solution, and then transferred to a T-75 culture flask. When the number of cells reaches 1X 10⁴To 2X 10⁴In this case, hybridoma cells were inoculated into 25ml culture flasks containing DMEM + 10% FBS +1 XHT at 37 ℃ and 5% CO₂Culturing under the condition. The cell status was monitored every two days with an inverted microscope to reach 75% confluence or 5X 10⁵When the cell/ml and the cell viability are over 85 percent, the next operation is carried out.

Example 3: production and purification of monoclonal antibodies

Cells were harvested from the flasks and the number of viable cells was determined. If the activity is higher than 80%, the cells are inoculated into roller bottles previously filled with 200mL of an antibody production medium (Hybridoma-SFM + 2.5% FBS (Low IgG)) at an initial cell density of 0.25X 10⁵cells/mL to 0.5X 10⁵cells/mL. After inoculation, the cells were incubated at 300r/h in a roller bottle incubator without CO₂Culturing at 37 ℃ for 14-16 days, and collecting the cell culture supernatant. Then, the cell suspension was transferred to a 350ml centrifuge flask and centrifuged at 3220 Xg at 4 ℃ for 15 minutes, followed by filtration with a 0.45 μm filter to remove cells and cell debris.

Cell culture supernatant was loaded onto a pre-equilibrated Protein a affinity column, which was then washed with 10CV of equilibration buffer (1x PBS, pH 7.2) until the OD of the flow-through sample became zero. The antibody was eluted from the column with 5CV of elution buffer (0.1M sodium citrate buffer, pH 3.0), with only one elution performed without a gradient. The eluted solution was collected in a clean tube and neutralized with neutralization buffer (1M Trizma base, pH 9.0) to a final pH of 7.0. The collected antibody was dialyzed overnight against 100 elution volumes of PBS (pH 7.4) at 2-8 ℃ with 3 buffer exchanges to ensure complete buffer exchange. The dialyzed antibody was transferred to a clean tube and sterile filtered in a biosafety cabinet using a 0.22 μm syringe filter to yield purified mAb002 antibody. Purified mAb002 antibody was aliquoted and stored at-20 ℃ until use.

Example 4: determination of concentration and purity of monoclonal antibody

The concentration of mAb002 antibody was determined by Nanodrop (thermoldisser, Nanodrop2000C spectrophotometer) at a280 nm. Table 1 lists the information relating to the antibodies and their concentrations.

TABLE 1

Antibodies	Isoforms	Concentration (mg/ml)	Immunogen	Animal # s
					mAb002	IgG2b,κ	0.6	Protein Complex B	SJL#7516,7517

The purity of the mAb002 antibody was determined by SDS-PAGE and SEC-HPLC as shown in FIGS. 1 and 2, respectively.

The test method is as follows:

SDS-PAGE: mu.g of antibody was mixed with 5. mu.L DTT (final concentration 50mM), 12.5. mu.L 4 Xloading buffer, and then adjusted to 50. mu.L with PBS. The samples were heated at 95 ℃ for 5 minutes and the prepared samples were pipetted into the NuPAGE gels. Electrophoresis was performed on Surelock Xcell at 100V for 2 hours. After electrophoresis, the gel was stained with i-Run quick stain solution and photographed with a CCD camera. Marker is 161-0373 from Bio-rad.

SEC-HPLC: mu.g of the antibody was injected onto a column (TSK gel G3000SWXL with an internal diameter of 7.8 mm. times.30 cm) and 1 XPBS buffer was used as the mobile phase. The column temperature was 30 ℃ and the flow rate was set to 0.8 ml/min. The purity of the antibody preparation was determined based on the peak area corresponding to the antibody.

The results show that the prepared mAb002 antibody has higher concentration and purity.

Example 5: sequencing of monoclonal antibodies

And extracting the genome of the mAb002 antibody, sequencing, storing in a DNA form, and performing subsequent exogenous expression and monoclonal antibody production. The amino acid sequences of the heavy chain variable region and the light chain variable region of mAb002 antibody are shown below (the underlined parts are VH CDR1-3 and VL CDR1-3 in that order):

mAb002-VH(SEQ ID NO：7)：

QVQLQQPGAELVRPGTSVKLSCKASGYTFISYWMHWVKQRPGQGLEWIGVIDPSDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARYYYGSSRYFDVWGTGTTVTVSS

mAb002-VL(SEQ ID NO：8)：

YIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKVLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSYTLPWTFGGGTKLEIK

example 6: determination of kinetic parameters

According to the biofilm interference technique (BLI), using FortebioThe kinetic parameters of the antibody are measured by a detection instrument.

mAb002, coupled to 2 μ G/ml, was conjugated via protein G biosensor, to different concentrations of antigen (AAVXL32.1 protein capsid) as shown in table 2, where the antigen was diluted with PBS and subsequently dissociated in PBS. The kinetic parameters of the mAb002 antibody are shown in table 2, and the results of the kinetic characterization parameters are shown in fig. 6.

TABLE 2 kinetic parameters of mAb002 antibody

Concentration (nM)	Response to	KD(M)	kon(1/Ms)	kdis(1/s)
					2.25	5.9147	<1.0E-12	3.26E+06	<1.0E-07
1.13	4.8269	<1.0E-12	3.26E+06	<1.0E-07
					0.5625	2.6765	<1.0E-12	3.26E+06	<1.0E-07
0.2813	1.401	<1.0E-12	3.26E+06	<1.0E-07
					0.1406	0.7013	<1.0E-12	3.26E+06	<1.0E-07
0.0703	0.3383	<1.0E-12	3.26E+06	<1.0E-07

K_DIs the dissociation constant; kon is the antigen-antibody binding rate; kdis the antigen-antibody dissociation rate; k_D＝kdis/kon。

The results show that the dissociation constant of the antibody and antigen is low, indicating that both have excellent affinity.

Example 7: sandwich ELISA detection of AAV protein capsid

Antibody-conjugated biotin

1-10mg/mL mAb002 antibody (IgG) was labeled with a 20-fold molar excess of sulfo-NHS-LC-biotin reagent (Thermofisiher, cat # 20217), yielding 4-6 biotin groups per antibody molecule. Adjusting the molar ratio of sulfo-NHS-LC-biotin to protein to be not less than 20: 1, mixing and reacting at room temperature for 1 h. The buffer was replaced with a desalting column to obtain biotin (biotin) -conjugated antibody, designated mAb 002-biotin. The concentration of the antibody was determined by Nanodrop (thermoldisser, Nanodrop2000C spectrophotometer) at a280 nm.

Sandwich ELISA

The AAVXL32.1 protein capsid was detected by ELISA. FIG. 4 shows a flow chart of a double antibody sandwich ELISA method. And coating the mAb002 antibody in a 96-hole micro-porous plate to prepare a solid phase carrier. Then, the antigen to be tested AAVXL32.1 protein capsid is added and allowed to bind to mAb 002. The antigen-antibody complex formed on the solid phase carrier is separated from other substances in the liquid by washing. Next, mAb002-biotin was added and reacted to bind also to the antigen to form a sandwich. After washing the unbound mAb002-biotin, streptavidin (thermoleisher, cat # N100) was labeled with horseradish peroxidase (HRP) to recognize the biotin group. After thorough washing again, TMB substrate (Thermofisiher, cat # 002023) was added for color development. TMB is converted to blue by HRP catalysis and to the final yellow by the action of an acid.

Thus, the antigen AAVXL32.1 protein capsid is qualitatively or quantitatively analyzed according to the color change and the shade. As shown in fig. 5, the absorbance (o.d. value) was measured at a wavelength of 450nm with a microplate reader, and the concentration of the antigen to be measured was obtained using a standard curve. The result shows that the lowest detection concentration (detection limit) of the detection method using the mAb002 antibody of the disclosure can reach 0.4ng/ml (4E7vg/ml), and the sensitivity is high; coefficient of correlation R²The accuracy is good, and is 0.9984.

While the present disclosure has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing is a more detailed description of the disclosure than is possible with reference to the specific embodiments, and that no limitation to the specific embodiments of the disclosure is intended. Various changes in form and detail, including simple deductions or substitutions, may be made by those skilled in the art without departing from the spirit and scope of the present disclosure.

Sequence listing

<110> belief medical science & technology (Suzhou) Co., Ltd

<120> detection method for AAV protein coat

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH CDR1

<400> 1

Ser Tyr Trp Met His

1 5

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH CDR2

<400> 2

Val Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 3

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH CDR3

<400> 3

Tyr Tyr Tyr Gly Ser Ser Arg Tyr Phe Asp Val

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL CDR1

<400> 4

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL CDR2

<400> 5

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL CDR3

<400> 6

Gln Gln Ser Tyr Thr Leu Pro Trp Thr

1 5

<210> 7

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mAb002-VH

<400> 7

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Tyr Gly Ser Ser Arg Tyr Phe Asp Val Trp Gly Thr

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 8

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mAb002-VL

<400> 8

Tyr Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Val Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Ser Tyr Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 9

<211> 737

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of AAVXL32.1

<400> 9

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ser Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn

210 215 220

Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn

260 265 270

His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr

405 410 415

Phe Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn

435 440 445

Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe

450 455 460

Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu

465 470 475 480

Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp

485 490 495

Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu

500 505 510

Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His

515 520 525

Lys Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe

530 535 540

Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met

545 550 555 560

Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu

565 570 575

Arg Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro

580 585 590

Ala Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp

595 600 605

Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro

610 615 620

His Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly

625 630 635 640

Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro

645 650 655

Ala Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile

660 665 670

Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu

675 680 685

Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser

690 695 700

Asn Tyr Ala Arg Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly

705 710 715 720

Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro

725 730 735

Leu