Method for detecting in-vitro relative potency of HPV vaccine or antigen liquid

Abstract

The invention relates to the use of enzyme-linked immunosorbent assay to detect the in vitro relative potency of HPV-VLP antigen stock solutions and vaccine finished products (types 6, 11, 16 and 18). The enzyme-linked immunosorbent assay has universality on detection of antigens produced by different expression systems (hansenula polymorpha, pichia pastoris, escherichia coli and insect cells), the standardization level of HPV vaccine quality evaluation is improved, and a foundation is laid for promoting research and development of high-quality HPV vaccines.

Description

Technical Field

[0001] The present invention relates to the field of detection, and in particular to an enzyme-linked immunosorbent assay method for detecting the in vitro relative efficacy of HPV vaccines or antigen stock solutions, especially the detection of quadrivalent (types 6, 11, 16 and 18) HPV vaccines. Background Art

[0002] Human papillomavirus (HPV) infects humans extensively and can cause malignant tumors such as cervical cancer, which seriously endangers human health. HPV vaccines can effectively prevent HPV infection and the diseases it causes. The World Health Organization is implementing a global plan to eliminate cervical cancer. HPV vaccines that have entered clinical research contain multiple antigens (2, 3, 4, 9, 11, 14, 15 valent) and use different expression systems (Saccharomyces cerevisiae, Hansenula, Pichia pastoris, Escherichia coli and insect cells).

[0003] In vitro efficacy experiments that have a good correlation with in vivo efficacy can replace in vivo efficacy experiments in mice for vaccine release testing, saving time and reducing animal use. In vitro relative efficacy is a key quality attribute of HPV vaccines, and quality control requires the determination of type-specific antigen content. At present, each manufacturer uses its own HPV type-specific monoclonal antibody to establish a double antibody sandwich ELISA method to detect its own L1 antigen, but the test results vary greatly and the standards are inconsistent; some research and development units lack advantageous monoclonal antibodies that recognize key type-specific epitopes, which limits the development and standardization of HPV vaccines. It is necessary to develop HPV type-specific monoclonal antibodies, establish a unified and standardized HPV type-specific antigen quantification method, improve the standardization level of HPV vaccine quality evaluation, and promote the development of high-quality HPV vaccines. Summary of the invention

[0004] On the one hand, the present invention provides an in vitro relative potency detection method for HPV vaccine or HPV antigen stock liquid, characterized in that it comprises the following steps: coating an ELISA plate with an HPV non-type specific antibody, adding a sample to be tested for incubation, and washing the plate; adding an enzyme-labeled HPV type-specific antibody for incubation, and washing the plate; adding a color developing solution for color development; adding a stop solution, and determining the OD value.

[0005] In some embodiments, the HPV non-type specific antibody comprises CDR-H1 as shown in SEQ ID NO: 25, CDR-H2 as shown in SEQ ID NO: 26, CDR-H3 as shown in SEQ ID NO: 27, CDR-L1 as shown in SEQ ID NO: 28, CDR-L2 as shown in SEQ ID NO: 29, and CDR-L3 as shown in SEQ ID NO: 30. In some embodiments, the method further comprises: detection of a standard, and the detection method of the standard is the same as that of the sample to be tested. In some embodiments, the method further comprises fitting a four-parameter curve Y=(da) / (1+(X / c)^b)+a according to the antigen concentration and OD value, wherein a is the lower limit of the asymptote, b is the slope of the linear interval line, c is the 50% reaction point of the curve (between a and d) (expressed as concentration), d is the upper limit of the asymptote, Y is the OD value, X is the antigen concentration, and the EC50 values ​​of the standard and the sample to be tested are obtained, and the in vitro relative potency of the sample to be tested = EC50 of the standard / EC50 of the sample to be tested. In some embodiments, the ELISA plate is prefabricated, and the sample to be tested is added to the prefabricated ELISA plate coated with HPV non-type specific antibodies for incubation, and the detection method does not include the step of using the HPV non-type specific antibody to coat the plate.

[0006] On the other hand, the present invention also provides an ELISA kit, the kit comprising an enzyme labeling plate coated with HPV type non-specific antibodies, the HPV type non-specific antibodies comprising CDR-H1 as shown in SEQ ID NO: 25, CDR-H2 as shown in SEQ ID NO: 26, CDR-H3 as shown in SEQ ID NO: 27, CDR-L1 as shown in SEQ ID NO: 28, CDR-L2 as shown in SEQ ID NO: 29, and CDR-L3 as shown in SEQ ID NO: 30. In some embodiments, the kit is used for in vitro relative potency detection of HPV vaccines or HPV antigen stock solutions, the kit further comprising a detection antibody, the detection antibody is an enzyme-labeled HPV type-specific antibody, wherein the HPV type-specific antibody is selected from HPV 6-specific antibody, HPV 11-specific antibody, HPV 16-specific antibody and / or HPV 18-specific antibody.

[0007] In some embodiments, the HPV vaccine or HPV antigen stock solution is HPV type 6, and the HPV type-specific antibody is an HPV antibody specific to HPV6, comprising CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6.

[0008] In some embodiments, the HPV vaccine or HPV antigen stock solution is HPV type 11, and the HPV type-specific antibody is an HPV antibody specific to HPV 11, comprising CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12.

[0009] In some embodiments, the HPV vaccine or HPV antigen stock solution is HPV16 type, and the HPV type-specific antibody is an HPV antibody specific to HPV 16, comprising CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18.

[0010] In some embodiments, the HPV vaccine or HPV antigen stock solution is HPV18 type, and the HPV type-specific antibody is an HPV antibody specific to HPV 18, comprising CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24.

[0011] In some embodiments, the HPV vaccine is a HPV6 or HPV11 2-valent vaccine, and the HPV type-specific antibodies are HPV 6-specific antibodies and HPV 11-specific antibodies.

[0012] The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6;

[0013] The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO:7, CDR-H2 as shown in SEQ ID NO:8, CDR-H3 as shown in SEQ ID NO:9, CDR-L1 as shown in SEQ ID NO:10, CDR-L2 as shown in SEQ ID NO:11, and CDR-L3 as shown in SEQ ID NO:12.

[0014] In some embodiments, the HPV vaccine is a HPV6 or HPV16 2-valent vaccine, and the HPV type-specific antibody is an HPV 6-specific antibody or an HPV 16-specific antibody.

[0015] The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6;

[0016] The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18.

[0017] In some embodiments, the HPV vaccine is a HPV6 or HPV18 2-valent vaccine, and the HPV type-specific antibodies are HPV 6-specific antibodies and HPV 18-specific antibodies.

[0018] The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6;

[0019] The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24.

[0020] In some embodiments, the HPV vaccine is a HPV16 or HPV18 2-valent vaccine, and the HPV type-specific antibodies are HPV 16-specific antibodies and HPV 18-specific antibodies.

[0021] The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18;

[0022] The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24.

[0023] In some embodiments, the HPV vaccine is an HPV16 or HPV11 2-valent vaccine, and the HPV type-specific antibodies are HPV 16-specific antibodies and HPV 11-specific antibodies.

[0024] The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18;

[0025] The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO:7, CDR-H2 as shown in SEQ ID NO:8, CDR-H3 as shown in SEQ ID NO:9, CDR-L1 as shown in SEQ ID NO:10, CDR-L2 as shown in SEQ ID NO:11, and CDR-L3 as shown in SEQ ID NO:12.

[0026] In some embodiments, the HPV vaccine is a HPV11 or HPV18 2-valent vaccine, and the HPV type-specific antibody is an HPV 11-specific antibody or an HPV 18-specific antibody.

[0027] The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12;

[0028] The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24.

[0029] In some embodiments, the HPV vaccine is a HPV6, HPV11, HPV16 trivalent vaccine, and the HPV type-specific antibodies are HPV 6-specific antibodies, HPV 11-specific antibodies, and HPV16-specific antibodies.

[0030] The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6;

[0031] The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12;

[0032] The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18.

[0033] In some embodiments, the HPV vaccine is a HPV6, HPV11, HPV18 trivalent vaccine, and the HPV type-specific antibodies are HPV 6-specific antibodies, HPV 11-specific antibodies, and HPV18-specific antibodies.

[0034] The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6;

[0035] The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12;

[0036] The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24.

[0037] In some embodiments, the HPV vaccine is a HPV6, HPV16, HPV18 trivalent vaccine, and the HPV type-specific antibodies are HPV 6-specific antibodies, HPV 16-specific antibodies, and HPV18-specific antibodies.

[0038] The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6;

[0039] The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18;

[0040] The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24.

[0041] In some embodiments, the HPV vaccine is a HPV11, HPV16, HPV18 trivalent vaccine, and the HPV type-specific antibodies are HPV 6-specific antibodies, HPV 16-specific antibodies, and HPV18-specific antibodies.

[0042] The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12;

[0043] The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18;

[0044] The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24.

[0045] In some embodiments, the HPV vaccine is an HPV 4-valent vaccine, and the HPV type-specific antibodies are HPV6-specific antibodies, HPV 11-specific antibodies, HPV 16-specific antibodies, and HPV 18-specific antibodies.

[0046] The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6;

[0047] The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12;

[0048] The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18;

[0049] The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24.

[0050] In some embodiments, the acceptance criteria for the method are as follows:

[0051] ① In the four-parameter fitting curve, the regression determination coefficient R is required 2 ≥0.98, four-parameter curve da≥2.0, the b value ratio of the sample curve to the reference curve is between 0.8-1.2;

[0052] ② The 10%-75% value of the upper and lower limits of OD is specified as the linear interval: OD10%=a+10%×(d–a), OD75%=a+75%×(d–a), and the number of linear effective concentration points within the dilution gradient of each sample is required to be ≥3; the CV between replicate wells in each linear interval of the sample is ≤30%;

[0053] ③ The OD value of the negative control well is ≤0.1, and the OD value of the positive control well is ≥0.8.

[0054] If any of the above criteria is not met, the test is invalid.

[0055] In some embodiments, the HPV type-specific antibody contained in the kit is selected from HPV 6-specific antibody, HPV 11-specific antibody, HPV 16-specific antibody and / or HPV 18-specific antibody, wherein:

[0056] The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6;

[0057] The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12;

[0058] The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18;

[0059] The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24.

[0060] In some embodiments, the HPV type non-specific antibody comprises VH as shown in SEQ ID NO:39 and VL as shown in SEQ ID NO:40.

[0061] In some embodiments, the HPV 6-specific antibody comprises the VH set forth in SEQ ID NO:31 and the VL set forth in SEQ ID NO:32.

[0062] In some embodiments, the HPV 11-specific antibody comprises the VH set forth in SEQ ID NO:33 and the VL set forth in SEQ ID NO:34.

[0063] In some embodiments, the HPV 16-specific antibody comprises the VH set forth in SEQ ID NO:35 and the VL set forth in SEQ ID NO:36.

[0064] In some embodiments, the HPV 18-specific antibody comprises the VH set forth in SEQ ID NO:37 and the VL set forth in SEQ ID NO:38.

[0065] In some embodiments, the HPV type-nonspecific antibody or type-specific antibody comprises: (1) a heavy chain constant region as shown in SEQ ID NO: 53, and (2) a light chain constant region as shown in any one of SEQ ID NO: 51 and SEQ ID NO: 52.

[0066] In some embodiments, the method is an in vitro relative efficacy detection method for HPV vaccines, further comprising a desorption step.

[0067] In some embodiments, the detection antibody is an enzyme-labeled detection antibody. In some embodiments, the enzyme-labeled detection antibody refers to a detection antibody labeled with horseradish peroxidase (Horseradish Peroxidase, HRP), alkaline phosphatase (AlkalinePhosphatase, AKP), β-galactosidase (β-Galactosidase), glucose oxidase (GlucoseOxidase, GOD), and acid phosphatase. In some embodiments, the enzyme-labeled detection antibody is an HPV type-specific antibody labeled with horseradish peroxidase. In some embodiments, the enzyme-labeled detection antibody is an HPV 6-specific antibody labeled with horseradish peroxidase. In some embodiments, the enzyme-labeled detection antibody is an HPV 11-specific antibody labeled with horseradish peroxidase. In some embodiments, the enzyme-labeled detection antibody is an HPV 16-specific antibody labeled with horseradish peroxidase. In some embodiments, the enzyme-labeled detection antibody is an HPV 18-specific antibody labeled with horseradish peroxidase.

[0068] In some embodiments, the kit further comprises a sample diluent, an enzyme-labeled antibody diluent, a color developing solution, a stop solution, and a washing solution.

[0069] In some embodiments, the detection method specifically comprises:

[0070] (1) Balance, equilibrate all reagents to room temperature;

[0071] (2) Prepare the solution by diluting the washing solution with distilled water or deionized water to the working concentration;

[0072] (3) Sample processing: pre-dilution of the standard and the sample to be tested, the Sa01 concentration after HPV6 pre-dilution is 5000-20000 ng / mL, the Sa01 concentration after HPV11 pre-dilution is 10000-20000 ng / mL, the Sa01 concentration after HPV16 pre-dilution is 2500-10000 ng / mL, and the Sa01 concentration after HPV18 pre-dilution is 5000-20000 ng / mL (the concentration marked on the sample to be tested is regarded as the initial concentration and diluted to the aforementioned target concentration), and then 2-fold or 3-fold serial dilution, a total of 6-11 dilutions;

[0073] (4) Add the diluted samples and positive and negative controls to the corresponding wells of the ELISA plate;

[0074] (5) Incubation: incubation in a 37°C constant temperature incubator;

[0075] (6) Washing: After the incubation is completed, carefully remove the sealing film, discard the liquid in the wells, add diluted washing solution, and wash the plate;

[0076] (7) Add enzyme, dilute the detection antibody to the working concentration with enzyme-labeled antibody diluent, and add the diluted detection antibody to each well;

[0077] (8) Incubation: incubation in a 37°C constant temperature incubator;

[0078] (9) Washing: After the incubation is completed, carefully remove the sealing film, discard the liquid in the wells, add diluted washing solution, and wash the plate;

[0079] (10) Color development: add color developing solution to each well, seal the plate with a sealing film, and incubate in a 37°C constant temperature incubator away from light;

[0080] (11) Stop / read: remove the sealing film, add stop solution to each well, mix well, and read (set the wavelength to 620 nm, and subtract the reading at 620 nm from the reading at 450 nm to correct for optical defects in the plate); (12) Data processing: fit a four-parameter curve Y=(da) / (1+(X / c)^b)+a based on the antigen concentration and OD value, where a is the lower limit of the asymptote, b is the slope of the linear interval line, c is the 50% reaction point of the curve (between a and d) (expressed as concentration), d is the upper limit of the asymptote, Y is the OD value, and X is the antigen concentration.

[0081] In some embodiments, the detection method further comprises: (13) calculating the in vitro relative potency, wherein the in vitro relative potency = EC50 of the standard substance / EC50 of the test sample.

[0082] Those skilled in the art will understand that the HPV type-specific antibody corresponds to the type of the vaccine or antigen stock solution to be tested. For example, when the vaccine or antigen stock solution to be tested is HPV 6 type, the HPV type-specific antibody is an HPV 6-specific antibody; when the vaccine or antigen stock solution to be tested is HPV 11 type, the HPV type-specific antibody is an HPV 11-specific antibody; when the vaccine to be tested is HPV 6 and HPV16 2-valent, the HPV type-specific antibody is an HPV 6-specific antibody and an HPV16-specific antibody; when the vaccine to be tested is HPV 6, HPV11, HPV16, and HPV18 4-valent, the HPV type-specific antibody is an HPV 6-specific antibody, an HPV11-specific antibody, an HPV16-specific antibody, and an HPV18-specific antibody.

[0083] Those skilled in the art will understand that when the sample to be tested is a multivalent vaccine (e.g., 2-valent, 3-valent, 4-valent), the detection antibodies corresponding to the HPV antigen types contained therein are respectively used for detection. For example, when the sample to be tested is a 4-valent vaccine of HPV 6, HPV11, HPV16, and HPV18, the detection antibodies specific to the HPV 6, HPV11, HPV16, and HPV18 types are respectively used for detection. The ELISA detection kit of the present invention can be a kit for detecting a single type of HPV antigen, such as a kit for detecting HPV 6, HPV11, HPV16, or HPV18; it can also be a kit for detecting a multivalent vaccine, such as a kit for detecting a 4-valent vaccine of HPV 6, HPV11, HPV16, and HPV18, and the kit contains 4 detection antibodies.

[0084] The method of the present invention has high accuracy, good precision, and high specificity, and is universal for detecting antigens produced by different expression systems. It improves the standardization level of HPV vaccine quality evaluation and lays the foundation for promoting the research and development of high-quality HPV vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0086] Figure 1 An exemplary diagram showing the layout of the ELISA plate used in the detection method of the present invention;

[0087] Figure 2A , Figure 2B , Figure 2C , Figure 2DThe test results of HPV 6, HPV 11, HPV 16, and HPV 18 antigen stock solutions of manufacturer 1 are displayed respectively;

[0088] Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E , Figure 3F The results of in vitro relative potency tests of four kinds of antigen stock solutions and finished vaccines (the finished quaternary vaccine has been desorbed) from six domestic HPV research and development units (Xiamen Wantai Canghai Biotechnology Co., Ltd. - Escherichia coli expression system, Chengdu Institute of Biological Products Co., Ltd. - Hansen yeast expression system, Jiangsu Ruike Biotechnology Co., Ltd. - Hansen yeast expression system, Shanghai Bowei Biotechnology Co., Ltd. - Hansen yeast expression system, Beijing Shenzhou Cell Biotechnology Group Co., Ltd. - Insect cell expression system, Shanghai Zerun Biotechnology Co., Ltd. - Pichia pastoris expression system) are shown respectively;

[0089] Figure 4A , Figure 4B , Figure 4C , Figure 4D The linear regression equations of the theoretical logarithmic values ​​of VLP and the theoretical logarithmic values ​​of potency of HPV 6, HPV 11, HPV 16, and HPV 18 are shown respectively; DETAILED DESCRIPTION

[0090] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0091] Description of amino acid and nucleic acid sequences

[0092]

[0093]

[0094]

[0095]

[0096] Example 1: Preparation of anti-HPV antibodies

[0097] The coding sequences of the antibody light and heavy chain variable regions were cloned into a eukaryotic expression vector carrying the human IgG1 constant region coding sequence, transiently transfected into CHO cells for secretory expression, and five anti-HPV antibody clones with a purity of >90% were obtained through affinity purification, namely F5-77, F5-187, F5-196, F5-203 and F5-127.

[0098] The amino acid sequences of VH, VL and CL of the antibodies are shown in Table 1.

[0099] Table 1: Amino acid sequences of VH, VL and CL of anti-HPV antibodies

[0100]

[0101]

[0102] Amino acid sequence of human IgG1 constant region:

[0103] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0104] Example 2: Characterization of anti-HPV antibodies - Characterization of antigen binding specificity by ELISA

[0105] HPV type 9 L1 proteins [HPV6 (genbank number: UNG35082.1), HPV11 (genbank number: AAA46935.1), HPV16 (genbank number: QGC89586.1), HPV18 (genbank number: ACU01871.1), HPV31 (genbank number: OP900721.1), HPV33 (genbank number: WAN40740.1), HPV45 (genbank number: AAY86494.1), HPV52 (genbank number: BBD06702.1) and HPV58 (genbank number: WAN40708.1)] diluted to 2 μg / mL with PBS were added to a 96-well ELISA plate (NEST, 504201) at 100 μL / well and coated overnight at 4°C. The solution was removed, washed twice with PBST, and blocked with blocking solution (PBS + 5% BSA) at 37°C for 2 hours. The solution was removed, and the antibody (concentration 1 μg / mL) diluted with diluent (PBS + 5% BSA) was added to the microplate at 100 μL / well and incubated at 37°C for 1 hour. The solution was removed, washed 3 times with PBST, and 100 μL of 1W-fold diluted mouse anti-human IgG Fc-HRP (Vazyme self-produced) was added to each well and incubated at 37°C for 1 hour. The solution was removed, washed 3 times with PBST, and 100 μL of chromogenic substrate TMB was added to each well, and incubated at 37°C for 10 minutes in the dark. The solution was removed, washed 3 times with PBST, and 50 μL of 2M sulfuric acid was added to each well. The OD value at 450nm was measured on a multifunctional microplate reader (Tecan, Spark). The results are shown in Table 2.

[0106] Table 2: Protein binding activity of anti-HPV antibodies

[0107]

[0108]

[0109] The results showed that F5-77, F5-187, F5-196 and F5-203 only bound to a single type of protein and were specific; F5-127 bound to all nine types of proteins and was a broad-spectrum binding antibody.

[0110] Example 3: ELISA method to detect the relative efficacy of quadrivalent (types 6, 11, 16 and 18) human papillomavirus vaccines in vitro

[0111] Material preparation

[0112] ELISA plate: dilute the antibody F5-127 prepared in Example 1 with a coating solution (0.05M carbonate buffer) to a protein content of 2μg / ml, add 100μL to each well of a 96-well ELISA plate, and place at 2-8°C for adsorption for 24 hours. Remove the coating solution and wash the plate three times with a coating wash solution (0.2mol / L PBST);

[0113] Enzyme labeling reagent: 5 mg / ml horseradish peroxidase-labeled F5-77, F5-187, F5-196, and F5-203;

[0114] Enzyme labeling reagent diluent: 0.1mol / LPBS, 0.05% Tween 20, 1% BSA, 0.1% P300 preservative

[0115] Colorimetric solution: TMB colorimetric solution (Incbiotech, catalog number EL0001)

[0116] Stop solution: Add 1M sulfuric acid to ultrapure water to make a solution with a concentration of 2 mol / L

[0117] Sample diluent: 0.1mol / LPBS, 0.05% Tween 20, 0.5% Casein, 0.1% P300 preservative

[0118] Concentrated washing solution: PBST: 0.2 mol / L, PBS with pH 7.4, with 0.05% Tween 20 added by volume

[0119] Positive control diluent: 0.1 mol / L PBS, 0.05% Tween 20, 0.5% Casein, 0.1% P300 preservative

[0120] Negative control: 0.1 mol / L PBS, 0.05% Tween 20, 0.5% Casein, 0.1% P300 preservative

[0121] Standards: recombinant HPV6-L1 protein, HPV11-L1 protein, HPV16-L1 protein, HPV18-L1 protein (manufacturer 1, E. coli)

[0122] Samples to be tested: Antigen stock solutions from manufacturer 1 (HPV6, HPV11, HPV16, HPV18 antigen stock solutions, 3 batches each).

[0123] Experimental methods

[0124] 1. Balance: Equilibrate all reagents to room temperature (at least 30 min) and mix the frozen samples.

[0125] 2. Liquid preparation: Dilute the concentrated detergent solution 20 times with distilled water.

[0126] 3. Sample processing: pre-dilution of the standard and the sample to be tested, the Sa01 concentration after HPV6 pre-dilution is 10000 ng / mL, the Sa01 concentration after HPV11 pre-dilution is 20000 ng / mL, the Sa01 concentration after HPV16 pre-dilution is 2500, and the Sa01 concentration after HPV18 pre-dilution is 10000 ng / mL (the concentration marked on the sample to be tested is regarded as the initial concentration, diluted to the aforementioned target concentration), and then 2-fold serial dilution, a total of 11 dilutions (01-11), 2 replicates for each dilution.

[0127] 4. Add sample: press Figure 1 Arrange the plates (one corresponding ELISA plate for each type), add 100 μL of the diluted standard or the diluted corresponding sample to be tested to the corresponding wells, and add 4 wells of positive and negative controls. For example, if the concentration gradient of the diluted HPV6 standard is 9.8-10000 ng / mL, the example plate layout is as follows: Figure 1 .

[0128] 5. Incubation: After sealing the plate with sealing film, place it in a 37℃ constant temperature incubator and incubate for 60 minutes.

[0129] 6. Washing: After incubation, carefully remove the sealing film, discard the liquid in the wells, add at least 300μL 1× washing solution to each well, let it stand for 30s and then discard the washing solution. Wash the plate 5 times in a row, and try to remove the residual liquid for the last time.

[0130] 7. Add enzyme: dilute the enzyme labeling reagent (100×) to 1× with enzyme labeling reagent diluent, and add 100 μL enzyme labeling reagent to each well.

[0131] 8. Incubation: After sealing the plate with sealing film, place it in a 37℃ constant temperature incubator and incubate for 60 minutes.

[0132] 9. Repeat step 6.

[0133] 10. Color development: Add 100 μL of color development solution to each well, seal the plate with a sealing film, and place it in a 37°C constant temperature incubator in the dark for 20 minutes.

[0134] 11. Stop / read: Carefully remove the sealing film, add 50 μL of stop solution to each well, mix gently, and then read the value. Set the wavelength to 620 nm and subtract the reading at 620 nm from the reading at 450 nm (to correct for optical defects in the plate).

[0135] 12. Data processing: Soft Max 4.8 (software provided by Perkin Elmer multifunctional microplate reader) was used to perform four-parameter curve fitting. The difference between the EC50 of the test sample and the EC50 of the standard was analyzed to evaluate the in vitro relative efficacy of the test sample. The in vitro relative efficacy = EC50 of the standard / EC50 of the test sample. The results are shown in Figure 2A , Figure 2B , Figure 2C , Figure 2D .

[0136] Four-parameter curve fitting equation: Y = (da) / (1+(X / c)^b)+a

[0137] a = asymptotic lower limit

[0138] b = slope of the linear interval line

[0139] c = 50% reaction point of the curve (between a and d) (expressed as concentration)

[0140] d = asymptotic upper limit

[0141] Y=OD value

[0142] X = concentration

[0143] 13. Experimental acceptance criteria:

[0144] ① In the four-parameter fitting curve, the regression determination coefficient R is required 2 ≥0.98, four-parameter curve da≥2.0, the b value ratio of the sample curve to the reference curve is between 0.8-1.2;

[0145] ② The 10%-75% value of the upper and lower limits of OD is specified as the linear interval: OD10%=a+10%×(d–a), OD75%=a+75%×(d–a), and the number of linear effective concentration points within the dilution gradient of each sample is required to be ≥3; the CV between replicate wells in each linear interval of the sample is ≤30%;

[0146] ③ The OD value of the negative control well is ≤0.1, and the OD value of the positive control well is ≥0.8.

[0147] Example 4: Specificity

[0148] The study is conducted on the addition of different HPV antigens to the HPV single-type antigen to be tested, that is, the ability of the type-specific detection antibody to detect the titers of the remaining three antigens after mixing four HPV antigens. The specificity is evaluated by calculating the test concentration / theoretical concentration ratio (%), that is, the relative bias (%). Taking 30% as the standard, the average relative bias of the four-type reagents meets this standard.

[0149] Detection method: Use the standard product and antigen stock solution of manufacturer 1 in Example 3, and perform detection by a method basically the same as Example 3 (the difference is that when each type of antigen is added to the test sample, an equal proportion of HPV6, HPV11, HPV16, and HPV18 antigen stock solutions are added).

[0150] The specificity results of HPV type 4 reagent testing are shown in Table 3-6.

[0151] Table 3 HPV6 type specificity

[0152]

[0153] Table 4 HPV11 type specificity

[0154]

[0155] Table 5 HPV16 type specificity

[0156]

[0157] Table 6 HPV18 type specificity

[0158]

[0159] Example 5: Universality of detection of antigens in different expression systems

[0160] The method of Example 3 was used to test four kinds of antigen stock solutions and finished vaccine products (the finished quaternary vaccine product was desorbed) from six domestic HPV research and development units (F Company - Xiamen Wantai Canghai Biotechnology Co., Ltd. - Escherichia coli expression system, C Company - Chengdu Institute of Biological Products Co., Ltd. - Hansen's yeast expression system, D Company - Jiangsu Ruike Biotechnology Co., Ltd. - Hansen's yeast expression system, B Company - Shanghai Bowei Biotechnology Co., Ltd. - Hansen's yeast expression system, E Company - Beijing Shenzhou Cell Biotechnology Group Co., Ltd. - Insect cell expression system, G Company - Shanghai Zerun Biotechnology Co., Ltd. - Pichia pastoris expression system), and the results are as follows: Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E , Figure 3F This method can effectively detect antigens from different companies, with a relative efficacy of 50%-200%.

[0161] Example 6: Standard Curve and Linearity

[0162] Acceptable standard: Plot the logarithm of the theoretical concentration value (abscissa) and the logarithm of the measured concentration value (ordinate), and use the least square method for linear regression. The correlation coefficient of the linear regression equation should not be less than 0.98. For the relative accuracy, intermediate precision and potency compliance level, the range should cover at least 80%-150% of the concentration level.

[0163] Verification results: Since there is no HPV-VLP standard, the vaccine manufacturer's antigen (the standard recombinant HPV6-L1 protein, HPV11-L1 protein, HPV16-L1 protein, and HPV18-L1 protein in Example 3) was used to determine its mass concentration, and its OD signal was detected after gradient dilution. The mass concentration was calculated by four-parameter fitting. Each dilution experiment was repeated twice, and the average value of the calculated concentration was taken for fitting. Specifically, the slope of the HPV6 type standard curve was 1.002, and R 2 The slope of the curve for HPV11 is 0.9874, R 2 The slope of the HPV16 curve is 0.9926, R 2 The slope of the HPV18 curve is 0.9889, R 2 is 0.9994( Figure 4A , Figure 4B , Figure 4C , Figure 4D The linear range of HPV6 was 5-3333 ng / ml, that of HPV11 was 14-3333 ng / ml, that of HPV16 was 5-3333 ng / ml, and that of HPV18 was 14-10000 ng / ml, indicating that the method had good detection performance.

[0164] Example 7: Relative Accuracy

[0165] Acceptable standard: The relative bias of each potency level relative to the potency measurement value should be within ±12%; the logarithm of the theoretical potency value (abscissa) is used to make a linear regression with the logarithm of the corresponding potency measurement value (ordinate), and the slope of the regression equation should be within ±12%. within the range.

[0166] Verification results: According to the method 9401 of the 2020 edition of the Chinese Pharmacopoeia and the calculation formula under the relative accuracy evaluation method in "I. Basic elements of method verification", the relative bias and confidence interval of the relative potency measurement value of each potency level were calculated. The results are shown in Tables 7-10. The relative biases were all within the range of ±12%. The logarithm of the theoretical potency value (abscissa) was used to make a linear regression on the logarithm of the corresponding potency measurement value (ordinate).

[0167] The relative bias of HPV6 at 1000ng / ml concentration among the four types of reagents exceeded the range of ±12%, and the average relative bias of the other types and concentrations met the requirements. The slope of the standard curve of HPV6 was 1.002, the slope of the standard curve of HPV11 was 0.9874, the slope of the standard curve of HPV16 was 0.9926, and the slope of the standard curve of HPV18 was 0.9889.

[0168] Table 7 Relative accuracy of HPV6 type

[0169]

[0170] Table 8 Relative accuracy of HPV11

[0171]

[0172] Table 9 Relative accuracy of HPV16 types

[0173]

[0174] Table 10 Relative accuracy of HPV18 types

[0175]

[0176] Example 8: Intermediate Precision

[0177] Acceptable standard: The geometric coefficient of variation (GCV, %) of the relative potency measurement value of each potency level should not be greater than 20%.

[0178] Verification results: The in vitro potency part of the Pharmacopoeia 2020 Edition IV 9401 Guidelines was calculated. The precision of the relative potency determination method is generally expressed by geometric standard deviation (GSD) or geometric coefficient of variation (GCV, %), and evaluated by analysis of variance (ANONA). The geometric standard deviation, geometric coefficient of variation and upper confidence limit of the relative potency determination value of each potency level were compared to see if they met the requirements. The results are shown in Tables 11-14. The geometric coefficient of variation of the relative potency determination values ​​of each potency level was less than 20%.

[0179] Among the four types of reagents, the GCV of HPV6 type was 21% when the test concentration was 1000, which was greater than 20%, and the other tests passed this standard.

[0180] Table 11 HPV6 intermediate precision

[0181] Potency level Number of experiments GSD GCV 1000 3 1.2 21％ 700 3 1.0 5％ 400 3 1.0 2％ 100 3 1.0 2％ 20 3 1.1 5％

[0182] Table 12 HPV11 intermediate precision

[0183] Potency level Number of experiments GSD GCV 1000 3 1.0 2％ 700 3 1.1 5％ 400 3 1.0 3％ 100 3 1.1 8％ 20 3 1.1 14％

[0184] Table 13 HPV16 intermediate precision

[0185] Potency level Number of experiments GSD GCV 1000 3 1.1 7％ 700 3 1.0 4％ 400 3 1.2 17％ 100 3 1.2 20％ 20 3 1.1 12％

[0186] Table 14 HPV18 intermediate precision

[0187] Potency level Number of experiments GSD GCV 1000 3 1.1 9％ 700 3 1.1 8％ 400 3 1.1 8％ 100 3 1.1 13％ 20 3 1.2 24％

Claims

1. A method for detecting the relative potency of an HPV vaccine or HPV antigen stock solution in vitro, characterized in that: The method comprises the following steps: coating an ELISA plate with HPV non-type specific antibodies, adding a sample to be tested for incubation, and washing the plate; adding an enzyme-labeled HPV type specific antibody for incubation, and washing the plate; adding a color developing solution for color development; and adding a stop solution to determine the OD value.

2. The method according to claim 1, wherein the HPV non-type specific antibody comprises CDR-H1 as shown in SEQ ID NO: 25, CDR-H2 as shown in SEQ ID NO: 26, CDR-H3 as shown in SEQ ID NO: 27, CDR-L1 as shown in SEQ ID NO: 28, CDR-L2 as shown in SEQ ID NO: 29, and CDR-L3 as shown in SEQ ID NO:

30.

3. An ELISA kit, comprising an ELISA plate coated with HPV type non-specific antibodies, wherein the HPV type non-specific antibodies comprise CDR-H1 as shown in SEQ ID NO: 25, CDR-H2 as shown in SEQ ID NO: 26, CDR-H3 as shown in SEQ ID NO: 27, CDR-L1 as shown in SEQ ID NO: 28, CDR-L2 as shown in SEQ ID NO: 29, and CDR-L3 as shown in SEQ ID NO:

30.

4. The method according to claim 1 further comprises fitting a four-parameter curve Y=(da) / (1+(X / c)^b)+a according to the antigen concentration and the OD value, wherein a is the lower limit of the asymptote, b is the slope of the linear interval line, c is the 50% reaction point of the curve (between a and d) (expressed as concentration), d is the upper limit of the asymptote, Y is the OD value, and X is the antigen concentration, to obtain the EC50 values ​​of the standard and the sample to be tested, and the in vitro relative efficacy of the sample to be tested=EC50 of the standard / EC50 of the sample to be tested.

5. The method according to claim 1, wherein the ELISA plate is prefabricated, and the sample to be tested is added to the prefabricated ELISA plate coated with HPV non-type specific antibodies and incubated, and the detection method does not include the step of using the plate coated with HPV non-type specific antibodies.

6. The kit according to claim 3, which is used for in vitro relative efficacy detection of HPV vaccines or HPV antigen stock solutions, the kit further comprising a detection antibody, which is an enzyme-labeled HPV type-specific antibody, wherein the HPV type-specific antibody is selected from HPV 6-specific antibody, HPV 11-specific antibody, HPV 16-specific antibody and / or HPV 18-specific antibody.

7. The detection method according to any one of claims 1-2, 4 and 5, (1) The HPV vaccine or HPV antigen stock solution is HPV type 6, and the HPV type-specific antibody is an HPV antibody specific for HPV6, comprising CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6; (2) The HPV vaccine or HPV antigen stock solution is HPV11 type, and the HPV type-specific antibody is an HPV antibody specific for HPV 11, comprising CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12; (3) The HPV vaccine or HPV antigen stock solution is HPV16 type, and the HPV type-specific antibody is an HPV 16-specific HPV antibody, comprising CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18; (4) The HPV vaccine or HPV antigen stock solution is HPV18 type, and the HPV type-specific antibody is an HPV antibody specific for HPV 18, comprising CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24; (5) The HPV vaccine is a HPV6 or HPV11 2-valent vaccine, and the HPV type-specific antibodies are HPV 6-specific antibodies and HPV 11-specific antibodies, The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6, The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12; (6) The HPV vaccine is a HPV6 or HPV16 2-valent vaccine, and the HPV type-specific antibodies are HPV 6-specific antibodies and HPV 16-specific antibodies, The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6, The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18; (7) The HPV vaccine is a HPV6 or HPV18 2-valent vaccine, and the HPV type-specific antibodies are HPV 6-specific antibodies and HPV 18-specific antibodies, The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6, The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24; (8) The HPV vaccine is a HPV16 or HPV18 2-valent vaccine, and the HPV type-specific antibodies are HPV 16-specific antibodies and HPV 18-specific antibodies, The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18, The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24; (9) The HPV vaccine is a HPV16 or HPV11 2-valent vaccine, and the HPV type-specific antibodies are HPV 16-specific antibodies and HPV 11-specific antibodies. The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18, The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12; (10) The HPV vaccine is a HPV11 or HPV18 2-valent vaccine, and the HPV type-specific antibodies are HPV 11-specific antibodies and HPV 18-specific antibodies. The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12, The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24; (11) The HPV vaccine is a trivalent vaccine of HPV6, HPV11, and HPV16, and the HPV type-specific antibodies are HPV 6-specific antibodies, HPV 11-specific antibodies, and HPV16-specific antibodies, The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6, The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12, The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18; (12) The HPV vaccine is a trivalent vaccine of HPV6, HPV11, and HPV18, and the HPV type-specific antibodies are HPV 6-specific antibodies, HPV 11-specific antibodies, and HPV18-specific antibodies. The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6, The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12, The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24; (13) The HPV vaccine is a trivalent vaccine of HPV6, HPV16, and HPV18, and the HPV type-specific antibodies are HPV 6-specific antibodies, HPV 16-specific antibodies, and HPV18-specific antibodies. The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6, The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18, The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24; (14) The HPV vaccine is a trivalent vaccine of HPV11, HPV16, and HPV18, and the HPV type-specific antibodies are HPV 6-specific antibodies, HPV 16-specific antibodies, and HPV 18-specific antibodies. The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12, The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18, The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24; or (15) The HPV vaccine is a HPV quadrivalent vaccine, and the HPV type-specific antibodies are HPV 6-specific antibodies, HPV 11-specific antibodies, HPV 16-specific antibodies, and HPV 18-specific antibodies. The HPV 6-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6, The HPV 11-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12, The HPV 16-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18, The HPV 18-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO:

24.

8. According to the method according to any one of claims 1-2, 4-5, and 7, the HPV type non-specific antibody comprises VH as shown in SEQ ID NO: 39 and VL as shown in SEQ ID NO:

40.

9. The kit according to claim 6 or the detection method according to claim 7, wherein: The HPV 6-specific antibody comprises a VH as shown in SEQ ID NO: 31 and a VL as shown in SEQ ID NO: 32; The HPV 11-specific antibody comprises a VH as shown in SEQ ID NO: 33 and a VL as shown in SEQ ID NO: 34; The HPV 16-specific antibody comprises a VH as shown in SEQ ID NO: 35 and a VL as shown in SEQ ID NO: 36; The HPV 18-specific antibody comprises VH as shown in SEQ ID NO: 37 and VL as shown in SEQ ID NO:

38.

10. The detection method or kit according to any one of claims 1 to 9, wherein the HPV type-nonspecific antibody or type-specific antibody comprises: (1) a heavy chain constant region as shown in SEQ ID NO: 53, and (2) a light chain constant region as shown in any one of SEQ ID NO: 51 and SEQ ID NO:

52.

11. The detection method according to claim 1 or the kit according to claim 6, wherein the enzyme-labeled HPV type-specific antibody is selected from HPV type-specific antibodies labeled with horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase or acid phosphatase, preferably HPV type-specific antibodies labeled with horseradish peroxidase.

12. The kit according to any one of claims 3, 6, 9-11, further comprising a sample diluent, an enzyme-labeled antibody diluent, a color developing solution, a stop solution and a washing solution.

13. The method according to any one of claims 1, 4, 5, 7-11, specifically comprising: (1) Balance, equilibrate all reagents to room temperature; (2) Prepare the solution by diluting the washing solution with distilled water or deionized water to the working concentration; (3) Sample processing: pre-dilution of the standard and the sample to be tested, the Sa01 concentration after HPV6 pre-dilution is 5000-20000 ng / mL, the Sa01 concentration after HPV11 pre-dilution is 10000-20000 ng / mL, the Sa01 concentration after HPV16 pre-dilution is 2500-10000 ng / mL, and the Sa01 concentration after HPV18 pre-dilution is 5000-20000 ng / mL (the concentration marked on the sample to be tested is regarded as the initial concentration and diluted to the aforementioned target concentration), and then 2-fold serial dilution, a total of 6-11 dilutions; (4) Add the diluted samples and positive and negative controls to the corresponding wells of the ELISA plate; (5) Incubation: incubation in a 37°C constant temperature incubator; (6) Washing: After the incubation is completed, carefully remove the sealing film, discard the liquid in the wells, add diluted washing solution, and wash the plate; (7) Add enzyme, dilute the detection antibody to the working concentration with enzyme-labeled antibody diluent, and add the diluted detection antibody to each well; (8) Incubation: incubation in a 37°C constant temperature incubator; (9) Washing: After the incubation is completed, carefully remove the sealing film, discard the liquid in the wells, add diluted washing solution, and wash the plate; (10) Color development: add color developing solution to each well, seal the plate with a sealing film, and incubate in a 37°C constant temperature incubator away from light; (11) Stop / read: remove the sealing film, add stop solution to each well, mix, and read the value (set the wavelength to 620 nm, and subtract the reading at 620 nm from the reading at 450 nm to correct for optical defects in the plate); (12) Data processing: fitting a four-parameter curve Y = (da) / (1+(X / c)^b)+a according to the antigen concentration and OD value, where a is the lower limit of the asymptote, b is the slope of the linear interval line, c is the 50% reaction point of the curve (between a and d) (expressed as concentration), d is the upper limit of the asymptote, Y is the OD value, and X is the antigen concentration.

14. The method according to any one of claims 1, 4, 5, 7-11, 13, wherein the acceptable criteria are as follows: ① In the four-parameter fitting curve, the regression determination coefficient R is required 2 ≥0.98, four-parameter curve da≥2.0, the b value ratio of the sample curve to the reference curve is between 0.8-1.2; ② The 10%-75% value of the upper and lower limits of OD is specified as the linear interval: OD10%=a+10%×(d–a), OD75%=a+75%×(d–a), and the number of linear effective concentration points within the dilution gradient of each sample is required to be ≥3; the CV between replicate wells in each linear interval of the sample is ≤30%; ③ The OD value of the negative control well is ≤0.1, and the OD value of the positive control well is ≥0.8.

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