A method for detecting the relative efficacy of HPV vaccines or antigens in liquid form

Abstract

The present application relates to the application of enzyme-linked immunoassay for detecting the in vitro relative potency of HPV-VLP antigen stock solution and vaccine finished product (types 6, 11, 16 and 18). The enzyme-linked immunoassay of the present application has universality for detecting antigens produced by different expression systems (Hansenula, Pichia, Escherichia coli and insect cells), and improves the standardization level of HPV vaccine quality evaluation, and lays a foundation for promoting the research and development of high-quality HPV vaccine.

Description

Technical Field

[0001] This invention relates to the field of detection, and more specifically to an enzyme-linked immunosorbent assay (ELISA) method for detecting the relative in vitro potency of HPV vaccines or antigen stock solutions, particularly for the detection of quadrivalent (types 6, 11, 16, and 18) HPV vaccines. Background Technology

[0002] Human papillomavirus (HPV) widely infects humans and can lead to malignant tumors such as cervical cancer, seriously endangering human health. HPV vaccines can effectively prevent HPV infection and the diseases it causes, and the World Health Organization is implementing a global plan to eliminate cervical cancer. HPV vaccines already in clinical trials contain multiple antigens (2, 3, 4, 9, 11, 14, and 15 valents) and utilize different expression systems (Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Escherichia coli, and insect cells).

[0003] In vitro potency assays, which correlate well with in vivo potency, can replace in vivo potency assays in mice for vaccine release testing, saving time and reducing animal use. In vitro relative potency is a key quality attribute of HPV vaccines, and quality control requires the determination of type-specific antigen content. Currently, manufacturers use their own HPV type-specific monoclonal antibodies to establish double-antibody sandwich ELISA methods to detect their respective L1 antigens, but the results vary significantly, and standards are inconsistent. Some research institutions lack dominant monoclonal antibodies that identify key type-specific epitopes, limiting the standardization of HPV vaccine development and testing. There is a need to develop HPV type-specific monoclonal antibodies, establish unified and standardized methods for quantifying HPV type-specific antigens, improve the standardization level of HPV vaccine quality evaluation, and promote the development of high-quality HPV vaccines. Summary of the Invention

[0004] On one hand, the present invention provides a method for detecting the relative potency of HPV vaccines or HPV antigens in liquid form, characterized by comprising the following steps: coating an enzyme-linked immunosorbent assay (ELISA) plate with HPV non-type-specific antibodies, adding the sample to be tested for incubation, and washing the plate; adding enzyme-labeled HPV type-specific antibodies for incubation, and washing the plate; adding a chromogenic solution for color development; adding a stop solution and measuring 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, wherein the detection method for the standard is the same as that for the sample to be tested. In some embodiments, the method further includes fitting 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 asymptotic range, b is the slope of the linear interval, c is the 50% reaction point of the curve (between a and d) (expressed as concentration), d is the upper limit of the asymptotic range, Y is the OD value, and X is the antigen concentration, to obtain the EC50 values ​​of the standard and the test sample, and the in vitro relative potency of the test sample = EC50 of the standard / EC50 of the test sample. In some embodiments, the ELISA plate is pre-made, and the test sample is added to the pre-made ELISA plate coated with HPV non-type-specific antibodies for incubation. The detection method does not include the step of coating the plate with HPV non-type-specific antibodies.

[0006] On the other hand, the present invention also provides an ELISA kit comprising an enzyme-labeled plate coated with HPV type nonspecific antibodies, wherein the HPV type nonspecific 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. In some embodiments, the kit is used for in vitro relative potency testing of HPV vaccines or HPV antigen stock solutions. The kit further comprises a detection antibody, which is an enzyme-labeled HPV type-specific antibody, wherein the HPV type-specific antibody is selected from HPV 6 specific antibodies, HPV 11 specific antibodies, HPV 16 specific antibodies, and / or HPV 18 specific antibodies.

[0007] In some embodiments, the HPV vaccine or HPV antigen stock solution is HPV type 6, and the HPV type-specific antibody is an HPV6-specific HPV antibody, 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 11-specific HPV antibody, 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 HPV type 16, 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.

[0010] In some embodiments, the HPV vaccine or HPV antigen stock solution is HPV type 18, and the HPV type-specific antibody is an HPV 18-specific HPV antibody, 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 implementations, the HPV vaccine is a 2-valent HPV6 / HPV1 vaccine, and the HPV type-specific antibodies are HPV6-specific antibodies and HPV11-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 implementations, the HPV vaccine is a 2-valent HPV6 / HPV16 vaccine, and the HPV type-specific antibodies are HPV6-specific antibodies and HPV16-specific antibodies.

[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 implementations, the HPV vaccine is a bivalent HPV6 / HPV18 vaccine, and the HPV type-specific antibodies are HPV6-specific antibodies and HPV18-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 implementations, the HPV vaccine is a bivalent HPV16 / HPV18 vaccine, and the HPV type-specific antibodies are HPV16-specific antibodies and HPV18-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 implementations, the HPV vaccine is a 2-valent HPV16 / HPV11 vaccine, and the HPV type-specific antibodies are HPV16-specific antibodies and HPV11-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 implementations, the HPV vaccine is a bivalent HPV11 / HPV18 vaccine, and the HPV type-specific antibodies are HPV11-specific antibodies and HPV18-specific antibodies.

[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 implementations, the HPV vaccine is a trivalent vaccine against HPV6, HPV11, and HPV16, and the HPV type-specific antibodies are HPV6-specific antibodies, HPV11-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 implementations, the HPV vaccine is a trivalent vaccine against HPV 6, HPV 11, and HPV 18, and the HPV type-specific antibodies are HPV 6-specific antibodies, HPV 11-specific antibodies, and HPV 18-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 implementations, the HPV vaccine is a trivalent vaccine containing HPV6, HPV16, and HPV18, and the HPV type-specific antibodies are HPV6-specific antibodies, HPV16-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 implementations, the HPV vaccine is a trivalent vaccine containing HPV11, HPV16, and HPV18, and the HPV type-specific antibodies are HPV6-specific antibodies, HPV16-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 implementations, the HPV vaccine is a quadrivalent HPV vaccine, and the HPV type-specific antibodies are HPV6-specific antibodies, HPV11-specific antibodies, HPV16-specific antibodies, and HPV18-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 implementations, the acceptable criteria for the method are as follows:

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

[0052] ② The linear range is defined as 10%-75% of the upper and lower limits of OD: OD10% = a + 10% × (d – a), OD75% = a + 75% × (d – a). Each sample is required to have ≥3 effective linear concentration points within the dilution gradient range; the CV between replicates in each linear range 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 standards are not met, the test is invalid.

[0055] In some embodiments, the HPV type-specific antibody included in the kit is selected from HPV 6-specific antibodies, HPV 11-specific antibodies, HPV 16-specific antibodies, and / or HPV 18-specific antibodies, 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 nonspecific 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 VH as shown in SEQ ID NO: 31 and VL as shown in SEQ ID NO: 32.

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

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

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

[0065] In some embodiments, the HPV type nonspecific antibody or type specific antibody includes, for example: (1) the heavy chain constant region shown in SEQ ID NO: 53, and (2) the light chain constant region shown in either SEQ ID NO: 51 or SEQ ID NO: 52.

[0066] In some implementations, the method is an in vitro relative potency test method for HPV vaccines, and further includes 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 (HRP), alkaline phosphatase (AKP), β-galactosidase, glucose oxidase (GOD), or acid phosphatase. In some embodiments, the enzyme-labeled detection antibody is a horseradish peroxidase-labeled HPV type-specific antibody. In some embodiments, the enzyme-labeled detection antibody is a horseradish peroxidase-labeled HPV 6-specific antibody. In some embodiments, the enzyme-labeled detection antibody is a horseradish peroxidase-labeled HPV 11-specific antibody. In some embodiments, the enzyme-labeled detection antibody is a horseradish peroxidase-labeled HPV 16-specific antibody. In some embodiments, the enzyme-labeled detection antibody is a horseradish peroxidase-labeled HPV 18-specific antibody.

[0068] In some embodiments, the kit further includes sample diluent, enzyme-labeled antibody diluent, chromogenic solution, stop solution, and washing solution.

[0069] In some implementations, the detection method specifically includes:

[0070] (1) Equilibrate all reagents to room temperature;

[0071] (2) Prepare the solution by diluting the washing solution with distilled or deionized water to the concentration to be used.

[0072] (3) Sample processing: Pre-dilution of the standard and the sample to be tested. The SaO1 concentration after pre-dilution of HPV6 is 5000-20000 ng / mL, the SaO1 concentration after pre-dilution of HPV11 is 10000-20000 ng / mL, the SaO1 concentration after pre-dilution of HPV16 is 2500-10000 ng / mL, and the SaO1 concentration after pre-dilution of HPV18 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). Then, serial dilution is performed by 2 or 3 times, for a total of 6-11 dilutions.

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

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

[0075] (6) Washing: After incubation, 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 dilution buffer, and add the diluted detection antibody to each well;

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

[0078] (9) Washing: After incubation, 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 development solution to each well, seal the plate with sealing film, and incubate in a constant temperature incubator at 37°C in the dark.

[0080] (11) Termination / Reading: Remove the sealing film, add the stop solution to each well, mix well, and read the value (set to a wavelength of 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, 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 implementations, the detection method further includes: (13) calculating the in vitro relative potency, where the in vitro relative potency = EC50 of the standard / 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 sample being tested. For example, when the vaccine or antigen sample being tested is HPV 6, the HPV type-specific antibody is an HPV 6 specific antibody; when the vaccine or antigen sample being tested is HPV 11, the HPV type-specific antibody is an HPV 11 specific antibody; when the vaccine being tested is HPV 6 or HPV 16 (2-valent), the HPV type-specific antibody is an HPV 6 specific antibody or an HPV 16 specific antibody; when the vaccine being tested is HPV 6, HPV 11, HPV 16, or HPV 18 (4-valent), the HPV type-specific antibody is an HPV 6 specific antibody, an HPV 11 specific antibody, an HPV 16 specific antibody, or an HPV 18 specific antibody.

[0083] Those skilled in the art will understand that when the sample to be tested is a multivalent vaccine (e.g., bivalent, trivalent, or quadrivalent), detection is performed using antibodies corresponding to the HPV antigen types contained therein. For example, when the sample to be tested is a quadrivalent HPV vaccine (HPV 6, HPV11, HPV16, HPV18), detection is performed using antibodies specific to HPV 6, HPV11, HPV16, and HPV18, respectively. The ELISA test kit of the present invention can be a kit for detecting a single HPV antigen type, such as a kit for HPV 6, HPV11, HPV16, or HPV18; or it can be a kit for detecting multivalent vaccines, such as a kit for detecting a quadrivalent HPV vaccine (HPV 6, HPV11, HPV16, HPV18), and the kit contains four detection antibodies.

[0084] The method of this invention has high accuracy, precision, and specificity, and is universal for antigen detection 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. Attached Figure Description

[0085] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0086] Figure 1 This diagram shows an example of the layout of the enzyme-labeled plate used in the detection method of the present invention.

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

[0088] Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E , Figure 3F The results show the in vitro relative potency of four antigen stock solutions and finished vaccines (quadrivalent vaccines after desorption treatment) from six domestic HPV research institutions (Xiamen Wantai Canghai Biotechnology Co., Ltd. - Escherichia coli expression system, Chengdu Institute of Biological Products Co., Ltd. - Hansenula polymorpha expression system, Jiangsu Ruike Biotechnology Co., Ltd. - Hansenula polymorpha expression system, Shanghai Bowei Biotechnology Co., Ltd. - Hansenula polymorpha expression system, Beijing Shenzhou Cell Biotechnology Group Co., Ltd. - Insect cell expression system, and Shanghai Zerun Biotechnology Co., Ltd. - Pichia pastoris expression system).

[0089] Figure 4A , Figure 4B , Figure 4C , Figure 4D The linear regression equations for the theoretical logarithmic values ​​and valence logarithmic values ​​of the four HPV types (6, 11, 16, and 18) are displayed respectively. Detailed Implementation

[0090] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 antibody

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

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

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

[0100]

[0101]

[0102] The amino acid sequence of the human IgG1 constant region:

[0103] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

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

[0105] HPV 9 type L1 protein [HPV6 (genbank: UNG35082.1), HPV11 (genbank: AAA46935.1), HPV16 (genbank: QGC89586.1), HPV18 (genbank: ACU01871.1), HPV31 (genbank: OP900721.1), HPV33 (genbank: WAN40740.1), HPV45 (genbank: AAY86494.1), HPV52 (genbank: BBD06702.1), and HPV58 (genbank: WAN40708.1)] diluted to 2 μg / mL with PBS was added to a 96-well microplate (NEST, 504201) at 100 μL / well and incubated overnight at 4°C. After removing the solution, wash twice with PBST and block with blocking buffer (PBS + 5% BSA) at 37°C for 2 hours. Remove the solution again, add 100 μL of antibody diluted with diluent (PBS + 5% BSA) to each well, and incubate at 37°C for 1 hour. Remove the solution again, wash three times with PBST, add 100 μL of 10,000-fold diluted mouse anti-human IgG Fc-HRP (Vazyme product) to each well, and incubate at 37°C for 1 hour. Remove the solution again, wash three times with PBST, add 100 μL of chromogenic substrate TMB to each well, and incubate at 37°C in the dark for 10 minutes. Remove the solution again, wash three times with PBST, and add 50 μL of 2M sulfuric acid to each well. Measure the OD value at 450 nm using a multi-functional 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 bound to only a single type of protein, exhibiting specificity; while F5-127 bound to all nine types of proteins, classifying it as a broad-spectrum binding antibody.

[0110] Example 3: ELISA assay for the relative in vitro potency of a quadrivalent (types 6, 11, 16, and 18) human papillomavirus vaccine

[0111] Material preparation

[0112] ELISA plate: Dilute antibody F5-127 prepared in Example 1 with coating buffer (0.05M carbonate buffer) to a protein concentration of 2 μg / ml. Add 100 μL to each well of a 96-well ELISA plate and incubate at 2-8℃ for 24 hours for adsorption. Discard the coating buffer and wash the plate three times with coating washing buffer (0.2 mol / L PBST).

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

[0114] Enzyme-labeled reagent dilution buffer: 0.1 mol / L PBS, 0.05% Tween 20, 1% BSA, 0.1% P300 preservative.

[0115] Developing solution: TMB developing solution (Ingenic Biotechnology, catalog number EL0001)

[0116] Termination solution: Prepare a 2 mol / L solution by adding 1 M sulfuric acid to ultrapure water.

[0117] Sample dilution buffer: 0.1 mol / L PBS, 0.05% Tween 20, 0.5% Casein, 0.1% P300 preservative.

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

[0119] Positive control dilution: 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] Standard products: Recombinant HPV6-L1 protein, HPV11-L1 protein, HPV16-L1 protein, HPV18-L1 protein (manufactured by Manufacturer 1, E. coli)

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

[0123] Experimental methods

[0124] 1. Equilibration: Equilibrate all reagents to room temperature (at least 30 minutes), and mix frozen samples thoroughly.

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

[0126] 3. Sample preparation: Pre-dilution of the standard and the test sample. The pre-dilution concentration of SaO1 for HPV6 is 10000 ng / mL, the pre-dilution concentration of SaO1 for HPV11 is 20000 ng / mL, the pre-dilution concentration of SaO1 for HPV16 is 2500 ng / mL, and the pre-dilution concentration of SaO1 for HPV18 is 10000 ng / mL (the concentration marked on the test sample is regarded as the initial concentration and diluted to the aforementioned target concentration). Then, serial dilution is performed by 2-fold, for a total of 11 dilutions (01-11), with 2 replicates for each dilution.

[0127] 4. Sample addition: Follow the instructions in the appendix. Figure 1 Arrange the plates (one plate for each HPV type). Add 100 μL of diluted standard or diluted sample to the corresponding well, and add 4 wells each for positive and negative controls. For example, if the concentration gradient of the diluted HPV6 standard is 9.8-10000 ng / mL, the plate layout is shown below. Figure 1 .

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

[0129] 6. Washing: After incubation, carefully remove the sealing film, discard the liquid in the wells, add at least 300 μL of 1× washing buffer to each well, let stand for 30 seconds, then discard the washing buffer. Wash the plate 5 times consecutively, removing as much residual liquid as possible on the last wash.

[0130] 7. Add enzyme: Dilute the enzyme-labeled reagent (100×) to 1× with enzyme-labeled reagent dilution buffer, and add 100μL of enzyme-labeled reagent to each well.

[0131] 8. Incubation: After sealing the plate with sealing film, incubate it in a 37℃ constant temperature incubator 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 sealing film, and incubate in a 37°C constant temperature incubator in the dark for 20 min.

[0134] 11. Termination / Reading: 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 (correcting optical defects in the calibration plate) from the reading at 450 nm.

[0135] 12. Data Processing: Soft Max version 4.8 (the software included with the Perkin Elmer multi-mode microplate reader) was used for 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 potency of the test sample. In vitro relative potency = standard EC50 / test sample EC50. 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 straight line in the linear interval

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

[0140] d = upper limit of asymptote

[0141] Y = OD value

[0142] X = concentration

[0143] 13. Acceptable experimental criteria:

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

[0145] ② The linear range is defined as 10%-75% of the upper and lower limits of OD: OD10% = a + 10% × (d – a), OD75% = a + 75% × (d – a). Each sample is required to have ≥3 effective linear concentration points within the dilution gradient range; the CV between replicates in each linear range 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] This study investigates the addition of different HPV types to a single HPV antigen for testing, specifically evaluating the ability of a type-specific antibody to detect the titers of the remaining three antigens when four HPV types are mixed. Specificity is assessed by calculating the ratio of test concentration to theoretical concentration (%), i.e., the relative bias (%). The average relative bias of the four types of reagents meets a standard of 30%.

[0149] Detection method: The standard and antigen stock solution from Manufacturer 1 in Example 3 were used for detection in the same way as in Example 3 (the difference is that when adding the test sample, the antigen stock solutions of HPV6, HPV11, HPV16 and HPV18 were mixed in equal proportions for each type of antigen).

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

[0151] Table 3 HPV6 specificity

[0152]

[0153] Table 4 HPV11 type specificity

[0154]

[0155] Table 5 HPV16 specificity

[0156]

[0157] Table 6 HPV18 specificity

[0158]

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

[0160] Using the method described in Example 3, four antigen stock solutions and finished vaccine products (quadrivalent vaccine products underwent desorption treatment) from six domestic HPV research institutions (Company F - Xiamen Wantai Canghai Biotechnology Co., Ltd. - Escherichia coli expression system; Company C - Chengdu Institute of Biological Products Co., Ltd. - Hansenula polymorpha expression system; Company D - Jiangsu Ruike Biotechnology Co., Ltd. - Hansenula polymorpha expression system; Company B - Shanghai Bowei Biotechnology Co., Ltd. - Hansenula polymorpha expression system; Company E - Beijing Shenzhou Cell Biotechnology Group Co., Ltd. - Insect cell expression system; Company G - Shanghai Zerun Biotechnology Co., Ltd. - Pichia pastoris expression system) were tested. 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 ranging from 50% to 200%.

[0161] Example 6: Standard Curve and Linearity

[0162] Acceptable criteria: Plot the logarithm of the theoretical concentration (x-axis) against the logarithm of the measured concentration (y-axis), and perform linear regression using the least squares method. The correlation coefficient of the linear regression equation should be no less than 0.98. For relative accuracy, intermediate precision, and potency at the concentration level of conformity, this range should cover at least 80%–150% of the concentration level.

[0163] Verification Results: Since no HPV-VLP standard was available, vaccine manufacturer antigens (recombinant HPV6-L1 protein, HPV11-L1 protein, HPV16-L1 protein, and HPV18-L1 protein, as specified in Example 3) were used to determine their mass concentrations. After serial dilution, their OD signals were measured, and the mass concentrations were calculated by backfitting the standard curve using four parameters. Each dilution experiment was repeated twice, and the average backfitted concentration was used for fitting. Specifically, the slope of the HPV6 standard curve was 1.002, and R0... 2 The slope of the standard curve for HPV11 is 0.998; the slope of the curve is 0.9874, R 2 The slope of the standard curve for HPV16 is 0.9984; the slope of the curve is 0.9926, R 2 The slope of the HPV18 standard curve is 0.9992; the slope of the curve is 0.9889, R 2 It is 0.9994 ( Figure 4A , Figure 4B , Figure 4C , Figure 4D The linear range for HPV6 is 5-3333 ng / ml, for HPV11 it is 14-3333 ng / ml, for HPV16 it is 5-3333 ng / ml, and for HPV18 it is 14-10000 ng / ml, indicating that this method has good detection performance.

[0164] Example 7: Relative Accuracy

[0165] Acceptable criteria: The relative bias of each valence level relative to the measured valence value should be within ±12%; a linear regression of the logarithm of the theoretical valence value (x-axis) to the corresponding logarithm of the measured valence value (y-axis) should have a slope within [missing information]. Within the range.

[0166] Validation Results: The relative bias and confidence interval of the relative potency measurements for each potency level were calculated according to Method 9401 of the Chinese Pharmacopoeia 2020 and the formula under "I. Basic Elements of Method Validation" in the section on relative accuracy evaluation. The results are shown in Table 7-10. The relative biases were all within ±12%. A linear regression was performed using the logarithm of the theoretical potency value (x-axis) and the logarithm of the corresponding potency measurement value (y-axis).

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

[0168] Table 7 Relative Accuracy of HPV6

[0169]

[0170] Table 8 Relative Accuracy of HPV11

[0171]

[0172] Table 9 Relative Accuracy of HPV16

[0173]

[0174] Table 10 Relative Accuracy of HPV18

[0175]

[0176] Example 8: Intermediate Precision

[0177] Acceptable standard: The geometrical coefficient of variation (GCV, %) of each valence level relative to the valence measurement should not exceed 20%.

[0178] Verification Results: Calculations were performed according to the in vitro potency section of Guideline 9401, Part IV, 2020 Pharmacopoeia. The precision of relative potency assays is generally expressed as geometrical standard deviation (GSD) or geometrical coefficient of variation (GCV, %), and evaluated using analysis of variance (ANONA). The geometrical standard deviation, geometrical coefficient of variation, and their upper confidence limits for each potency level's relative potency assays were compared to ensure compliance with requirements. The results are shown in Tables 11-14. The geometrical coefficient of variation for each potency level's relative potency assays was less than 20%.

[0179] Among the four reagents, HPV6 showed a GCV of 21% at a test concentration of 1000, which is greater than 20%, and all other tests passed this standard.

[0180] Table 11 Intermediate Precision of HPV6

[0181] valence 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 Intermediate Precision of HPV11

[0183] valence 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 Intermediate Precision of HPV16

[0185] valence 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 Intermediate Precision of HPV18

[0187] valence 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 in vitro relative potency testing of an HPV vaccine or an HPV antigen bulk, characterized in that, comprises the steps of: coating an enzyme-labeled plate with HPV non-type specific antibody, adding the sample to be tested for incubation, washing the plate; adding enzyme-labeled HPV type specific antibody for incubation, washing the plate; adding color developing solution for color development; adding stop solution for determination of OD value, 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: DTS, and CDR-L3 as shown in SEQ ID NO:

30.

2. An ELISA kit comprising an enzyme-labeled plate coated with HPV non-type specific antibody, 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: DTS, and CDR-L3 as shown in SEQ ID NO:

30.

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

4. The method of claim 1, wherein the enzyme-labeled plate is pre-made, the pre-made enzyme-labeled plate coated with HPV non-type specific antibody is added to the sample to be tested for incubation, and the detection method does not comprise the step of coating the plate with HPV non-type specific antibody.

5. The kit of claim 2, for use in in vitro relative potency detection of HPV vaccine or HPV antigen stock solution, further comprising detection antibody, wherein the detection antibody is enzyme-labeled HPV type specific antibody, and 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.

6. The detection method of any one of claims 1, 3, 4, (1) the HPV vaccine or HPV antigen stock is HPV type 6, the HPV type-specific antibody is an HPV6-specific antibody, 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: KAS, and CDR-L3 as shown in SEQ ID NO: 6; (2) the HPV vaccine or HPV antigen stock is HPV type 11, the HPV type-specific antibody is an HPV 11-specific antibody, 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: WAS, and CDR-L3 as shown in SEQ ID NO: 12; (3) the HPV vaccine or HPV antigen stock is HPV type 16, the HPV type-specific antibody is an HPV 16-specific 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: SAS, and CDR-L3 as shown in SEQ ID NO: 18; (4) the HPV vaccine or HPV antigen stock is HPV type 18, the HPV type-specific antibody is an HPV 18-specific antibody, 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: RNN, and CDR-L3 as shown in SEQ ID NO: 24; (5) the HPV vaccine is a HPV6, HPV11 bivalent vaccine, the HPV type-specific antibody is an HPV 6-specific antibody, an HPV 11-specific antibody, 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: KAS, 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: WAS, and CDR-L3 as shown in SEQ ID NO:

12. the HPV 11-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 7, CDR-H2 as set forth in SEQ ID NO: 8, CDR-H3 as set forth in SEQ ID NO: 9, CDR-L1 as set forth in SEQ ID NO: 10, CDR-L2 as set forth in SEQ ID NO: 11: WAS, and CDR-L3 as set forth in SEQ ID NO: 12; (6) the HPV vaccine is a HPV6, HPV16 bivalent vaccine, the HPV type-specific antibody is a HPV 6-specific antibody, a HPV 16-specific antibody, the HPV 6-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 1, CDR-H2 as set forth in SEQ ID NO: 2, CDR-H3 as set forth in SEQ ID NO: 3, CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5: KAS, and CDR-L3 as set forth in SEQ ID NO: 6, the HPV 16-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 13, CDR-H2 as set forth in SEQ ID NO: 14, CDR-H3 as set forth in SEQ ID NO: 15, CDR-L1 as set forth in SEQ ID NO: 16, CDR-L2 as set forth in SEQ ID NO: 17: SAS, and CDR-L3 as set forth in SEQ ID NO: 18; (7) the HPV vaccine is a HPV6, HPV18 bivalent vaccine, the HPV type-specific antibody is a HPV 6-specific antibody, a HPV 18-specific antibody, the HPV 6-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 1, CDR-H2 as set forth in SEQ ID NO: 2, CDR-H3 as set forth in SEQ ID NO: 3, CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5: KAS, and CDR-L3 as set forth in SEQ ID NO: 6, the HPV 18-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 19, CDR-H2 as set forth in SEQ ID NO: 20, CDR-H3 as set forth in SEQ ID NO: 21, CDR-L1 as set forth in SEQ ID NO: 22, CDR-L2 as set forth in SEQ ID NO: 23: RNN, and CDR-L3 as set forth in SEQ ID NO: 24; (8) the HPV vaccine is a HPV16, HPV18 bivalent vaccine, the HPV type-specific antibody is a HPV 16-specific antibody, a HPV 18-specific antibody, 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: SAS, 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: RNN, and CDR-L3 as shown in SEQ ID NO: 24; (9) the HPV vaccine is an HPV16, HPV11 bivalent vaccine, the HPV type-specific antibody is an HPV 16-specific antibody, an HPV 11-specific antibody, 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: SAS, 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: WAS, and CDR-L3 as shown in SEQ ID NO: 12; (10) the HPV vaccine is an HPV11, HPV18 bivalent vaccine, the HPV type-specific antibody is an HPV 11-specific antibody, an HPV 18-specific antibody, 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: WAS, and CDR-L3 as shown in SEQ ID NO: 12, the HPV 18-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 19, CDR-H2 as set forth in SEQ ID NO: 20, CDR-H3 as set forth in SEQ ID NO: 21, CDR-L1 as set forth in SEQ ID NO: 22, CDR-L2 as set forth in SEQ ID NO: 23: RNN, and CDR-L3 as set forth in SEQ ID NO: 24; (11) the HPV vaccine is a HPV6, HPV11, HPV16 3-valent vaccine, and the HPV type-specific antibody is a HPV 6-specific antibody, a HPV 11-specific antibody, and a HPV 16-specific antibody, the HPV 6-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 1, CDR-H2 as set forth in SEQ ID NO: 2, CDR-H3 as set forth in SEQ ID NO: 3, CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5: KAS, and CDR-L3 as set forth in SEQ ID NO: 6, the HPV 11-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 7, CDR-H2 as set forth in SEQ ID NO: 8, CDR-H3 as set forth in SEQ ID NO: 9, CDR-L1 as set forth in SEQ ID NO: 10, CDR-L2 as set forth in SEQ ID NO: 11: WAS, and CDR-L3 as set forth in SEQ ID NO: 12, the HPV 16-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 13, CDR-H2 as set forth in SEQ ID NO: 14, CDR-H3 as set forth in SEQ ID NO: 15, CDR-L1 as set forth in SEQ ID NO: 16, CDR-L2 as set forth in SEQ ID NO: 17: SAS, and CDR-L3 as set forth in SEQ ID NO: 18; (12) the HPV vaccine is a HPV6, HPV11, HPV18 3-valent vaccine, and the HPV type-specific antibody is a HPV 6-specific antibody, a HPV 11-specific antibody, and a HPV 18-specific antibody, the HPV 6-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 1, CDR-H2 as set forth in SEQ ID NO: 2, CDR-H3 as set forth in SEQ ID NO: 3, CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5: KAS, and CDR-L3 as set forth in SEQ ID NO: 6, the HPV 11-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 7, CDR-H2 as set forth in SEQ ID NO: 8, CDR-H3 as set forth in SEQ ID NO: 9, CDR-L1 as set forth in SEQ ID NO: 10, CDR-L2 as set forth in SEQ ID NO: 11: WAS, and CDR-L3 as set forth in SEQ ID NO: 12, the HPV 11-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 7, CDR-H2 as set forth in SEQ ID NO: 8, CDR-H3 as set forth in SEQ ID NO: 9, CDR-L1 as set forth in SEQ ID NO: 10, CDR-L2 as set forth in SEQ ID NO: 11: WAS, and CDR-L3 as set forth in SEQ ID NO: 12, the HPV 18-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 19, CDR-H2 as set forth in SEQ ID NO: 20, CDR-H3 as set forth in SEQ ID NO: 21, CDR-L1 as set forth in SEQ ID NO: 22, CDR-L2 as set forth in SEQ ID NO: 23: RNN, and CDR-L3 as set forth in SEQ ID NO: 24; (13) the HPV vaccine is a HPV6, HPV16, HPV18 3-valent vaccine, and the HPV type-specific antibody is a HPV 6-specific antibody, a HPV 16-specific antibody, a HPV18-specific antibody, the HPV 6-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 1, CDR-H2 as set forth in SEQ ID NO: 2, CDR-H3 as set forth in SEQ ID NO: 3, CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5: KAS, and CDR-L3 as set forth in SEQ ID NO: 6, the HPV 16-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 13, CDR-H2 as set forth in SEQ ID NO: 14, CDR-H3 as set forth in SEQ ID NO: 15, CDR-L1 as set forth in SEQ ID NO: 16, CDR-L2 as set forth in SEQ ID NO: 17: SAS, and CDR-L3 as set forth in SEQ ID NO: 18, the HPV 18-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 19, CDR-H2 as set forth in SEQ ID NO: 20, CDR-H3 as set forth in SEQ ID NO: 21, CDR-L1 as set forth in SEQ ID NO: 22, CDR-L2 as set forth in SEQ ID NO: 23: RNN, and CDR-L3 as set forth in SEQ ID NO: 24; (14) the HPV vaccine is a HPV11, HPV16, HPV18 3-valent vaccine, and the HPV type-specific antibody is a HPV 11-specific antibody, a HPV 16-specific antibody, a HPV18-specific antibody, the HPV 6-specific antibody comprises CDR-H1 as set forth in SEQ ID NO: 1, CDR-H2 as set forth in SEQ ID NO: 2, CDR-H3 as set forth in SEQ ID NO: 3, CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5: KAS, and CDR-L3 as set forth in SEQ ID NO: 6, the HPV 11 -specific antibody comprises a CDR-H1 as set forth in SEQ ID NO: 7, a CDR-H2 as set forth in SEQ ID NO: 8, a CDR-H3 as set forth in SEQ ID NO: 9, a CDR-L1 as set forth in SEQ ID NO: 10, a CDR-L2 as set forth in SEQ ID NO: 11 : WAS, and a CDR-L3 as set forth in SEQ ID NO: 12, the HPV 16 -specific antibody comprises a CDR-H1 as set forth in SEQ ID NO: 13, a CDR-H2 as set forth in SEQ ID NO: 14, a CDR-H3 as set forth in SEQ ID NO: 15, a CDR-L1 as set forth in SEQ ID NO: 16, a CDR-L2 as set forth in SEQ ID NO: 17: SAS, and a CDR-L3 as set forth in SEQ ID NO: 18, the HPV 18 -specific antibody comprises a CDR-H1 as set forth in SEQ ID NO: 19, a CDR-H2 as set forth in SEQ ID NO: 20, a CDR-H3 as set forth in SEQ ID NO: 21, a CDR-L1 as set forth in SEQ ID NO: 22, a CDR-L2 as set forth in SEQ ID NO: 23: RNN, and a CDR-L3 as set forth in SEQ ID NO: 24; or (15) the HPV vaccine is an HPV 4-valent vaccine, and the HPV type-specific antibodies are an HPV 6 -specific antibody, an HPV 11 -specific antibody, an HPV 16 -specific antibody, an HPV 18 -specific antibody, the HPV 6 -specific antibody comprises a CDR-H1 as set forth in SEQ ID NO: 1, a CDR-H2 as set forth in SEQ ID NO: 2, a CDR-H3 as set forth in SEQ ID NO: 3, a CDR-L1 as set forth in SEQ ID NO: 4, a CDR-L2 as set forth in SEQ ID NO: 5: KAS, and a CDR-L3 as set forth in SEQ ID NO: 6, the HPV 11 -specific antibody comprises a CDR-H1 as set forth in SEQ ID NO: 7, a CDR-H2 as set forth in SEQ ID NO: 8, a CDR-H3 as set forth in SEQ ID NO: 9, a CDR-L1 as set forth in SEQ ID NO: 10, a CDR-L2 as set forth in SEQ ID NO: 11 : WAS, and a CDR-L3 as set forth in SEQ ID NO: 12, the HPV 11 -specific antibody comprises a CDR-H1 as set forth in SEQ ID NO: 7, a CDR-H2 as set forth in SEQ ID NO: 8, a CDR-H3 as set forth in SEQ ID NO: 9, a CDR-L1 as set forth in SEQ ID NO: 10, a CDR-L2 as set forth in SEQ ID NO: 11 : WAS, and a CDR-L3 as set forth in SEQ ID NO: 12, the HPV 18-specific antibody comprises a CDR-H1 as set forth in SEQ ID NO: 19, a CDR-H2 as set forth in SEQ ID NO: 20, a CDR-H3 as set forth in SEQ ID NO: 21, a CDR-L1 as set forth in SEQ ID NO: 22, a CDR-L2 as set forth in SEQ ID NO: 23: RNN, and a CDR-L3 as set forth in SEQ ID NO:

24. the HPV 18-specific antibody comprises a CDR-H1 as set forth in SEQ ID NO: 19, a CDR-H2 as set forth in SEQ ID NO: 20, a CDR-H3 as set forth in SEQ ID NO: 21, a CDR-L1 as set forth in SEQ ID NO: 22, a CDR-L2 as set forth in SEQ ID NO: 23: RNN, and a CDR-L3 as set forth in SEQ ID NO:

24.

7. The method of claim 6, wherein the HPV non-type specific antibody comprises a VH as set forth in SEQ ID NO: 39 and a VL as set forth in SEQ ID NO:

40.

8. The kit of claim 5, wherein, the HPV 6-specific antibody comprises a VH as set forth in SEQ ID NO: 31 and a VL as set forth in SEQ ID NO: 32; the HPV 11-specific antibody comprises a VH as set forth in SEQ ID NO: 33 and a VL as set forth in SEQ ID NO: 34; the HPV 16-specific antibody comprises a VH as set forth in SEQ ID NO: 35 and a VL as set forth in SEQ ID NO: 36; the HPV 18-specific antibody comprises a VH as set forth in SEQ ID NO: 37 and a VL as set forth in SEQ ID NO:

38.

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

38.

10. The detection method of claim 1 or the kit of claim 5, wherein the HPV non-type specific antibody or the HPV type specific antibody comprises (1) a heavy chain constant region as set forth in SEQ ID NO: 53, and (2) a light chain constant region as set forth in any one of SEQ ID NO: 51, SEQ ID NO:

52.

11. The detection method of claim 1 or the kit of claim 5, wherein the enzyme-labeled HPV type-specific antibody is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase or acid phosphatase-labeled HPV type-specific antibody.

12. The detection method or kit of claim 11, wherein the enzyme-labeled HPV type-specific antibody is horseradish peroxidase-labeled HPV type-specific antibody.

13. The kit of claim 5, further comprising sample diluent, enzyme-labeled antibody diluent, color developing solution, termination solution and washing solution.

14. The method of claim 6, specifically comprising: (1) equilibration, equilibrating all reagents to room temperature; (2) solution preparation, diluting the washing solution to the use concentration with distilled water or deionized water; (3) sample processing, pre-diluting the standard and the sample to be tested, the pre-diluted concentration of HPV6 is 5000-20000 ng / mL, the pre-diluted concentration of HPV11 is 10000-20000 ng / mL, the pre-diluted concentration of HPV16 is 2500-10000 ng / mL, the pre-diluted concentration of HPV18 is 5000-20000 ng / mL, the concentration marked on the sample to be tested is regarded as the initial concentration, which is diluted to the aforementioned target concentration, and then 2-fold serial dilution is performed, a total of 6-11 dilution degrees; (4) sample addition, adding the diluted sample and positive and negative controls into the corresponding wells of the enzyme-labeled plate; (5) incubation, incubating in a 37°C constant temperature incubator; (6) washing, after the incubation is completed, carefully removing the sealing plate film, discarding the liquid in the wells, adding the diluted washing solution and washing the plate; (7) enzyme addition, diluting the detection antibody to the use concentration with the enzyme-labeled antibody diluent, and adding the diluted detection antibody into each well; (8) incubation, incubating in a 37°C constant temperature incubator; (9) washing, after the incubation is completed, carefully removing the sealing plate film, discarding the liquid in the wells, adding the diluted washing solution and washing the plate; (10) color development, adding the color developing solution into each well, sealing the plate with the sealing plate film and incubating in a 37°C constant temperature incubator in the dark; (11) termination / reading, removing the sealing plate film, adding the termination solution into each well, mixing, reading, setting the wavelength to 620 nm, and subtracting the reading at 620 nm from the reading at 450 nm to correct the optical defects in the plate; (12) data processing, fitting the four-parameter curve Y = (d-a) / (1 + (X / c)^b) + a, wherein a is the lower limit of the asymptote, b is the linear interval straight line slope, c is the 50% reaction point of the curve, d is the upper limit of the asymptote, Y is the OD value, and X is the antigen concentration.

15. The method of claim 14, which can accept the following standards: ① Four-parameter fitting curve, the regression determination coefficient R 2 ≥0.98, four-parameter curve d-a≥2.0, the ratio of b value of sample curve to reference curve is between 0.8-1.2; ② The 10%-75% values of the upper and lower limits of OD are the linear interval: OD10% = a +10%×(d-a), OD75% = a +75%×(d-a), and the linear effective concentration points in the sample dilution gradient range are required to be ≥3; the CV between the duplicate wells in each linear interval of the sample is ≤30%. ③The OD value of the negative control hole is less than or equal to 0.1, and the OD value of the positive control hole is greater than or equal to 0.8.

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