A method for detecting the in vitro relative potency of a nine-type hpv vaccine or antigen bulk

Abstract

The present application relates to a method for detecting the in vitro relative potency of HPV-9 vaccine or antigen stock solution, and specifically applying enzyme-linked immunoassay to detect the in vitro relative potency of HPV-VLP antigen stock solution and vaccine finished product (types 6, 11, 16, 18, 31, 33, 45, 52 and 58). The enzyme-linked immunoassay of the present application has excellent accuracy and precision, improves the standardization level of HPV vaccine quality evaluation, and is expected to lay 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, specifically to an enzyme-linked immunosorbent assay (ELISA) method for detecting the relative in vitro efficacy of HPV vaccines or antigen stock solutions, particularly for the detection of nine-valent (types 6, 11, 16, 18, 31, 33, 45, 52, and 58) 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. HPV vaccines that have entered clinical trials contain multiple antigens (2, 3, 4, 9, 11, 14, and 15 valents).

[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, this invention provides a method for detecting the relative potency of HPV vaccines or HPV antigens in liquid form, characterized by 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. In some embodiments, the method further includes: detection of a standard, the detection method of which is the same as that of 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 curve, 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 curve, Y is the OD value, and X is the antigen concentration, obtaining the EC50 values ​​of the standard and the sample to be tested, and the relative potency of the sample to be tested = EC50 of the sample to be tested / EC50 of the standard. In some implementations, the ELISA plate is pre-made, and the sample to be tested is added to the pre-made ELISA plate coated with HPV non-type-specific antibodies and incubated. The detection method does not include the step of using HPV non-type-specific antibodies to coat the plate.

[0005] On the other hand, the present invention also provides a method for detecting the relative potency of a 9-valent HPV vaccine or HPV antigen in liquid form, characterized by comprising the following steps: adding the sample to be tested to an enzyme-labeled plate coated with HPV non-type-specific antibodies, incubating, and washing the plate; adding enzyme-labeled HPV type-specific antibodies, incubating, and washing the plate; adding a chromogenic solution, and developing color; adding a stop solution, and measuring the OD value; 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 curve, 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 curve, Y is the OD value, X is the antigen concentration, obtaining the EC50 values ​​of the standard and the sample to be tested, and the relative potency of the sample to be tested = EC50 of the sample to be tested / EC50 of the standard;

[0006] The HPV type-specific antibodies include HPV 6 specific antibodies, HPV 11 specific antibodies, HPV 16 specific antibodies, HPV 18 specific antibodies, HPV 31 specific antibodies, HPV 33 specific antibodies, HPV 45 specific antibodies, HPV 52 specific antibodies, and HPV 58 specific antibodies.

[0007] On the other hand, the present invention also provides an ELISA kit comprising an enzyme-labeled plate coated with HPV non-type-specific antibodies. In some embodiments, the kit is used for in vitro relative potency assays 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, HPV 18-specific antibodies, HPV 31-specific antibodies, HPV 33-specific antibodies, HPV 45-specific antibodies, HPV 52-specific antibodies, and / or HPV 58-specific antibodies.

[0008] In some embodiments, the 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.

[0009] In some embodiments, the HPV antigen stock solution is HPV11 type, and the HPV type-specific antibody is an HPV11-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.

[0010] In some embodiments, the HPV antigen stock solution is HPV16, and the HPV type-specific antibody is an HPV16-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.

[0011] In some embodiments, the HPV antigen stock solution is HPV18, and the HPV type-specific antibody is an HPV18-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.

[0012] In some embodiments, the HPV antigen stock solution is HPV31, and the HPV type-specific antibody is an HPV31-specific HPV antibody, 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.

[0013] In some embodiments, the HPV antigen stock solution is HPV33, and the HPV type-specific antibody is an HPV33-specific HPV antibody, comprising CDR-H1 as shown in SEQ ID NO: 31, CDR-H2 as shown in SEQ ID NO: 32, CDR-H3 as shown in SEQ ID NO: 33, CDR-L1 as shown in SEQ ID NO: 34, CDR-L2 as shown in SEQ ID NO: 35, and CDR-L3 as shown in SEQ ID NO: 36.

[0014] In some embodiments, the HPV antigen stock solution is HPV45, and the HPV type-specific antibody is an HPV45-specific HPV antibody, comprising CDR-H1 as shown in SEQ ID NO: 37, CDR-H2 as shown in SEQ ID NO: 38, CDR-H3 as shown in SEQ ID NO: 39, CDR-L1 as shown in SEQ ID NO: 40, CDR-L2 as shown in SEQ ID NO: 41, and CDR-L3 as shown in SEQ ID NO: 42.

[0015] In some embodiments, the HPV antigen stock solution is HPV52 type, and the HPV type-specific antibody is an HPV52-specific HPV antibody, comprising CDR-H1 as shown in SEQ ID NO: 43, CDR-H2 as shown in SEQ ID NO: 44, CDR-H3 as shown in SEQ ID NO: 45, CDR-L1 as shown in SEQ ID NO: 46, CDR-L2 as shown in SEQ ID NO: 47, and CDR-L3 as shown in SEQ ID NO: 48.

[0016] In some embodiments, the HPV antigen stock solution is HPV58, and the HPV type-specific antibody is an HPV58-specific HPV antibody, comprising CDR-H1 as shown in SEQ ID NO: 49, CDR-H2 as shown in SEQ ID NO: 50, CDR-H3 as shown in SEQ ID NO: 51, CDR-L1 as shown in SEQ ID NO: 52, CDR-L2 as shown in SEQ ID NO: 53, and CDR-L3 as shown in SEQ ID NO: 54.

[0017] In some implementations, the HPV vaccine is a 9-valent HPV vaccine, and the HPV type-specific antibodies are HPV6-specific antibodies, HPV11-specific antibodies, HPV16-specific antibodies, HPV18-specific antibodies, HPV31-specific antibodies, HPV33-specific antibodies, HPV45-specific antibodies, HPV52-specific antibodies, and HPV58-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 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.

[0020] 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;

[0021] 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;

[0022] The HPV 31 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;

[0023] The HPV 33 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 31, CDR-H2 as shown in SEQ ID NO: 32, CDR-H3 as shown in SEQ ID NO: 33, CDR-L1 as shown in SEQ ID NO: 34, CDR-L2 as shown in SEQ ID NO: 35, and CDR-L3 as shown in SEQ ID NO: 36;

[0024] The HPV 45 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 37, CDR-H2 as shown in SEQ ID NO: 38, CDR-H3 as shown in SEQ ID NO: 39, CDR-L1 as shown in SEQ ID NO: 40, CDR-L2 as shown in SEQ ID NO: 41, and CDR-L3 as shown in SEQ ID NO: 42;

[0025] The HPV 52 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 43, CDR-H2 as shown in SEQ ID NO: 44, CDR-H3 as shown in SEQ ID NO: 45, CDR-L1 as shown in SEQ ID NO: 46, CDR-L2 as shown in SEQ ID NO: 47, and CDR-L3 as shown in SEQ ID NO: 48;

[0026] The HPV 58 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 49, CDR-H2 as shown in SEQ ID NO: 50, CDR-H3 as shown in SEQ ID NO: 51, CDR-L1 as shown in SEQ ID NO: 52, CDR-L2 as shown in SEQ ID NO: 53, and CDR-L3 as shown in SEQ ID NO: 54.

[0027] In some implementations, the acceptable criteria for the method are as follows:

[0028] 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;

[0029] 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%.

[0030] ③ The OD value of the negative control well is ≤0.1, and the OD value of the positive control well is ≥0.8.

[0031] If any of the above standards are not met, the test is invalid.

[0032] In some embodiments, the HPV type-specific antibody is selected from HPV 6 specific antibody, HPV 11 specific antibody, HPV 16 specific antibody, HPV 18 specific antibody, HPV 31 specific antibody, HPV 33 specific antibody, HPV 45 specific antibody, HPV 52 specific antibody and / or HPV 58 specific antibody, wherein,

[0033] 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;

[0034] 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.

[0035] 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;

[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] The HPV 31 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;

[0038] The HPV 33 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 31, CDR-H2 as shown in SEQ ID NO: 32, CDR-H3 as shown in SEQ ID NO: 33, CDR-L1 as shown in SEQ ID NO: 34, CDR-L2 as shown in SEQ ID NO: 35, and CDR-L3 as shown in SEQ ID NO: 36;

[0039] The HPV 45 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 37, CDR-H2 as shown in SEQ ID NO: 38, CDR-H3 as shown in SEQ ID NO: 39, CDR-L1 as shown in SEQ ID NO: 40, CDR-L2 as shown in SEQ ID NO: 41, and CDR-L3 as shown in SEQ ID NO: 42;

[0040] The HPV 52 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 43, CDR-H2 as shown in SEQ ID NO: 44, CDR-H3 as shown in SEQ ID NO: 45, CDR-L1 as shown in SEQ ID NO: 46, CDR-L2 as shown in SEQ ID NO: 47, and CDR-L3 as shown in SEQ ID NO: 48;

[0041] The HPV 58 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 49, CDR-H2 as shown in SEQ ID NO: 50, CDR-H3 as shown in SEQ ID NO: 51, CDR-L1 as shown in SEQ ID NO: 52, CDR-L2 as shown in SEQ ID NO: 53, and CDR-L3 as shown in SEQ ID NO: 54.

[0042] In some embodiments, the HPV 6-specific antibody comprises VH as shown in SEQ ID NO: 55 and VL as shown in SEQ ID NO: 56.

[0043] In some embodiments, the HPV 11-specific antibody comprises VH as shown in SEQ ID NO: 57 and VL as shown in SEQ ID NO: 58.

[0044] In some embodiments, the HPV 16-specific antibody comprises VH as shown in SEQ ID NO: 59 and VL as shown in SEQ ID NO: 60.

[0045] In some embodiments, the HPV 18-specific antibody comprises VH as shown in SEQ ID NO: 61 and VL as shown in SEQ ID NO: 62.

[0046] In some embodiments, the HPV 31-specific antibody comprises VH as shown in SEQ ID NO: 63 and VL as shown in SEQ ID NO: 64.

[0047] In some embodiments, the HPV 33-specific antibody comprises VH as shown in SEQ ID NO: 65 and VL as shown in SEQ ID NO: 66.

[0048] In some embodiments, the HPV 45-specific antibody comprises VH as shown in SEQ ID NO: 67 and VL as shown in SEQ ID NO: 68.

[0049] In some embodiments, the HPV 52-specific antibody comprises VH as shown in SEQ ID NO: 69 and VL as shown in SEQ ID NO: 70.

[0050] In some embodiments, the HPV 58-specific antibody comprises VH as shown in SEQ ID NO: 71 and VL as shown in SEQ ID NO: 72.

[0051] In some embodiments, the HPV type-specific antibody comprises, for example: (1) a heavy chain constant region as shown in SEQ ID NO: 93, and (2) a light chain constant region as shown in either SEQ ID NO: 91 or SEQ ID NO: 92.

[0052] A polynucleotide encoding the type-specific antibody of the present invention. In some embodiments, the polynucleotide is selected from: (1) SEQ ID NO: 73 and 74; (2) SEQ ID NO: 75 and 76; (3) SEQ ID NO: 77 and 78; (4) SEQ ID NO: 79 and 80; (5) SEQ ID NO: 81 and 82; (6) SEQ ID NO: 83 and 84; (7) SEQ ID NO: 85 and 86; (8) SEQ ID NO: 87 and 88; (9) SEQ ID NO: 89 and 90.

[0053] In some implementations, the method is an in vitro relative potency test method for HPV vaccines, and further includes a desorption step.

[0054] 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. In some embodiments, the enzyme-labeled detection antibody is a horseradish peroxidase-labeled HPV 31-specific antibody. In some embodiments, the enzyme-labeled detection antibody is a horseradish peroxidase-labeled HPV 33-specific antibody. In some embodiments, the enzyme-labeled detection antibody is a horseradish peroxidase-labeled HPV 45-specific antibody. In some embodiments, the enzyme-labeled detection antibody is a horseradish peroxidase-labeled HPV 52-specific antibody. In some embodiments, the enzyme-labeled detection antibody is a horseradish peroxidase-labeled HPV 58-specific antibody.

[0055] In some embodiments, the kit further includes sample diluent, enzyme-labeled antibody diluent, chromogenic solution, stop solution, and washing solution.

[0056] In some implementations, the detection method specifically includes:

[0057] (1) Equilibrate all reagents to room temperature;

[0058] (2) Prepare the solution by diluting the washing solution with distilled or deionized water to the concentration to be used.

[0059] (3) Sample preparation: Pre-dilution of the standards and test samples. The pre-diluted SaO1 concentrations were 5000-20000 ng / mL for HPV6, 10000-20000 ng / mL for HPV11, 2500-10000 ng / mL for HPV16, 5000-20000 ng / mL for HPV18, and 5000-20000 ng / mL for HPV31. / mL, the pre-diluted SaO1 concentration of HPV33 is 5000-20000 ng / mL, the pre-diluted SaO1 concentration of HPV45 is 5000-20000 ng / mL, the pre-diluted SaO1 concentration of HPV52 is 5000-20000 ng / mL, the pre-diluted SaO1 concentration of HPV58 is 5000-20000 ng / mL (the concentration marked on the sample to be tested is regarded as the initial concentration, and it is diluted to the aforementioned target concentration), and then serially diluted 2-fold or 3-fold, for a total of 6-11 dilutions;

[0060] (4) Add the diluted sample and positive and negative controls to the corresponding wells of the ELISA plate;

[0061] (5) Incubation: Incubate at 37°C in a constant temperature incubator;

[0062] (6) Washing: After incubation, carefully remove the sealing film, discard the liquid in the wells, add diluted washing solution, and wash the plate.

[0063] (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;

[0064] (8) Incubation: Incubate at 37°C in a constant temperature incubator;

[0065] (9) Washing: After incubation, carefully remove the sealing film, discard the liquid in the wells, add diluted washing solution, and wash the plate.

[0066] (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.

[0067] (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.

[0068] In some implementations, the detection method further includes: (13) calculating the in vitro relative potency, where the in vitro relative potency = EC50 of the test sample / EC50 of the standard.

[0069] Those skilled in the art will understand that HPV type-specific antibodies correspond to the type of the vaccine or antigen sample being tested. For example, when the vaccine or antigen sample to be tested is HPV type 6, the HPV type-specific antibody is an HPV 6 specific antibody; when the vaccine or antigen sample to be tested is HPV type 11, the HPV type-specific antibody is an HPV 11 specific antibody; when the vaccine to be 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 to be 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; when the vaccine to be tested is HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 45, HPV 52, or HPV 58 (9-valent), the HPV type-specific antibody is an HPV 6 specific antibody. 6. Specific antibodies, HPV11 specific antibody, HPV16 specific antibody, HPV18 specific antibody, HPV31 specific antibody, HPV33 specific antibody, HPV45 specific antibody, HPV52 specific antibody, HPV58 specific antibody.

[0070] Those skilled in the art will understand that when the sample to be tested is a multivalent vaccine (e.g., 9-valent), detection is performed using antibodies corresponding to the HPV antigen types contained therein. For example, when the sample to be tested is a 9-valent vaccine (HPV 6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58), detection is performed using antibodies specific to HPV 6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58, respectively. The ELISA test kit of the present invention can be a kit for detecting a single type of HPV antigen, such as a kit for HPV 6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58; or it can be a kit for detecting multivalent vaccines, such as a kit for detecting nine-valent vaccines including HPV 6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58, and the kit contains nine detection antibodies.

[0071] The method of this invention has high accuracy, high precision, and high specificity, which 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

[0072] 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.

[0073] Figure 1 This diagram shows an example of the layout of the enzyme-labeled plate used in the detection method of the present invention.

[0074] Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E , Figure 2F , Figure 2G , Figure 2H , Figure 2I The results of the HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 45, HPV 52, and HPV 58 antigen stock solutions from Manufacturer 1 are displayed respectively.

[0075] Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E , Figure 3F , Figure 3G , Figure 3H , Figure 3I The linear regression equations for the logarithmic values ​​of the VLP theory and the logarithmic values ​​of the valence theory for HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 45, HPV 52, and HPV 58 are displayed respectively. Detailed Implementation

[0076] 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.

[0077] In this invention, peripheral blood was collected from 14 adult volunteers one month after completing the HPV 9-valent vaccine. The upper plasma layer and the middle PBMCs were obtained by density gradient centrifugation. Memory B cells specifically binding to the proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 were isolated from the PBMCs by flow cytometry using fluorescently labeled HPV type L1 proteins. Nested PCR was used to obtain transfectible PCR fragments with expression activity, which were then transfected into CHO cells for expression. Cell supernatants containing secreted antibodies were obtained, and binding activity was screened by ELISA, yielding 2475 binding-positive clones. Among them, 90 clones specifically bound to HPV type 6 L1 protein, and 181 clones specifically bound to HPV type 11 L1 protein. Specific binding to the L1 protein of HPV types was observed. 130 clones specifically bound to the L1 protein of HPV type 16, 97 clones to the L1 protein of HPV type 18, 94 clones to the L1 protein of HPV type 31, 87 clones to the L1 protein of HPV type 33, 88 clones to the L1 protein of HPV type 45, 90 clones to the L1 protein of HPV type 52, and 79 clones to the L1 protein of HPV type 58; 281 clones bound to the L1 protein of at least two types. Based on the binding and pseudovirus neutralization results in the cell supernatant, 25, 20, 35, 44, 42, 37, 26, 40, 23, and 63 recombinant antibodies were constructed from the above 10 types of antibodies, respectively. Based on the binding activity of each recombinant antibody to the corresponding HPV type protein and its neutralizing activity with each type of pseudovirus, the following 10 types of antibodies were ultimately selected: anti-HPV6 antibody F5-222, anti-HPV11 antibody F5-422, anti-HPV16 antibody F5-194, anti-HPV18 antibody F5-212, anti-HPV31 antibody F5-183, anti-HPV33 antibody F5-155, anti-HPV45 antibody F5-160, anti-HPV52 antibody F5-398, and anti-HPV58 antibody F5-354. All type-specific antibodies specifically bound to and neutralized the corresponding type protein or pseudovirus, with binding activity EC50 all below 40 ng / mL and neutralizing activity IC50 between 0.25 and 51.60 ng / mL.

[0078] Table 1. Description of amino acid and nucleic acid sequences

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Example 1: Preparation of anti-HPV antibody

[0086] 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. Nine anti-HPV antibody clones with a purity >90% were obtained through affinity purification, namely F5-222, F5-422, F5-194, F5-212, F5-183, F5-155, F5-160, F5-398, and F5-354.

[0087] The amino acid sequences of the antibody’s VH, VL, and CL are shown in Table 1.

[0088] The amino acid sequence of the human IgG1 constant region:

[0089] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0090] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK

[0091] SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE

[0092] DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG

[0093] KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

[0094] LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

[0095] QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0096] Example 2: Characterization of anti-HPV antibodies—characterizing antigen binding specificity by ELISA

[0097] 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.

[0098] Table 2: Protein binding activity of anti-HPV antibodies

[0099]

[0100] The results showed that F5-222, F5-422, F5-194, F5-212, F5-183, F5-155, F5-160, F5-398, and F5-354 bind to only a single type of protein, exhibiting specificity.

[0101] Example 3: ELISA assay for the in vitro relative potency of a nine-valent (types 6, 11, 16, 18, 31, 33, 45, 52, 58) human papillomavirus vaccine

[0102] Material preparation

[0103] Microplate: Three types of non-type-specific antibodies prepared in Example 1 were added to coating buffer (0.05M carbonate buffer) in equal proportions, resulting in a total protein content of 2 μg / ml. 100 μL was added to each well of a 96-well microplate and incubated at 2-8°C for 24 hours for adsorption. The coating buffer was then removed, and the plate was washed three times with coating wash buffer (0.2 mol / L PBST).

[0104] Enzyme-labeled reagents: 5 mg / ml horseradish peroxidase labeled F5-222, F5-422, F5-194, F5-212, F5-183, F5-155, F5-160, F5-398, F5-354;

[0105] Enzyme-labeled reagent dilution buffer: 0.1 mol / L PBS, 0.05% Tween 20, 1% BSA, 0.1% P300 preservative.

[0106] Developing solution: TMB developing solution (Ingenic Biotechnology, catalog number EL0001)

[0107] Termination solution: Prepare a 2 mol / L solution by adding 1 M sulfuric acid to ultrapure water.

[0108] Sample dilution buffer: 0.1 mol / L PBS, 0.05% Tween 20, 0.5% Casein, 0.1% P300 preservative.

[0109] Concentrated washing buffer: PBST: 0.2 mol / L, pH 7.4, with 0.05% Tween 20 added by volume.

[0110] Positive control dilution: 0.1 mol / L PBS, 0.05% Tween 20, 0.5% Casein, 0.1% P300 preservative

[0111] Negative control: 0.1 mol / L PBS, 0.05% Tween 20, 0.5% Casein, 0.1% P300 preservative

[0112] Standard products: Recombinant HPV6-L1 protein, HPV11-L1 protein, HPV16-L1 protein, HPV18-L1 protein, HPV31-L1 protein, HPV33-L1 protein, HPV45-L1 protein, HPV52-L1 protein, HPV58-L1 protein (manufactured in-house by Manufacturer 1, E. coli)

[0113] Samples to be tested: antigen stock solutions from manufacturer 1 (HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58 antigen stock solutions, 3 batches each).

[0114] Experimental methods

[0115] 1. Equilibration: Equilibrate all reagents to room temperature (at least 30 minutes), and mix frozen samples thoroughly.

[0116] 2. Solution preparation: Dilute the concentrated washing solution with distilled water 20 times.

[0117] 3. Sample Preparation: Pre-dilute the standards and test samples. The pre-diluted SaO1 concentrations are: HPV6 5000-10000 ng / mL; HPV11 10000-20000 ng / mL; HPV16 2500-10000 ng / mL; HPV18 5000-20000 ng / mL; HPV31 10000-20000 ng / mL; and HPV... 33. After pre-dilution, the SaO1 concentration was 10000-20000 ng / mL; after pre-dilution, the SaO1 concentration of HPV45 was 1000-20000 ng / mL; after pre-dilution, the SaO1 concentration of HPV52 was 1000-10000 ng / mL; after pre-dilution, the SaO1 concentration of HPV58 was 1000-10000 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, it was serially diluted 2-fold, for a total of 11 dilutions (01-11), with 2 replicates for each dilution.

[0118] 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 .

[0119] 5. Incubation: After sealing the plate with sealing film, place it in a 37℃ constant temperature incubator for 30 minutes.

[0120] 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.

[0121] 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.

[0122] 8. Incubation: After sealing the plate with sealing film, place it in a 37℃ constant temperature incubator for 30 minutes.

[0123] 9. Repeat step 6.

[0124] 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 15 min.

[0125] 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.

[0126] 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. The in vitro relative potency was calculated as: test sample EC50 / standard EC50. Results are shown in […]. Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E , Figure 2F , Figure 2G , Figure 2H , Figure 2I .

[0127] Four-parameter curve fitting equation: Y=(da) / (1+(X / c)^b)+a

[0128] a = asymptotic lower limit

[0129] b = slope of the straight line in the linear interval

[0130] c = 50% reaction point on the curve (between a and d) (expressed as concentration)

[0131] d = upper limit of asymptote

[0132] Y = OD value

[0133] X = concentration

[0134] 13. Acceptable experimental criteria:

[0135] 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;

[0136] 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%.

[0137] ③ The OD value of the negative control well is ≤0.1, and the OD value of the positive control well is ≥0.8.

[0138] Example 4: Specificity

[0139] This study investigates the addition of different HPV type antigens to a single HPV type antigen for testing, specifically evaluating the ability of a type-specific antibody to detect the titers of the remaining eight antigens when nine HPV type antigens 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 nine-type reagent meets a standard of 30%.

[0140] 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 sample to be tested, an equal proportion of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58 antigen stock solutions were added to each type of antigen).

[0141] The specificity results of HPV type 9 reagent detection are shown in Table 3-11.

[0142] Table 3 HPV6 specificity

[0143]

[0144] Table 4 HPV11 type specificity

[0145]

[0146] Table 5 HPV16 specificity

[0147]

[0148] Table 6 HPV18 specificity

[0149]

[0150] Table 7 HPV31 Model Specificity

[0151]

[0152] Table 8 HPV33 Model Specificity

[0153]

[0154] Table 9 HPV45 Model Specificity

[0155]

[0156] Table 10 HPV52 Model Specificity

[0157]

[0158] Table 11 HPV58 Model Specificity

[0159]

[0160] Example 5: Standard Curve and Linearity

[0161] 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.

[0162] Verification Results: Since no HPV-VLP standard was available, vaccine manufacturer 1 antigen (the standard recombinant HPV6-L1 protein, HPV11-L1 protein, HPV16-L1 protein, HPV18-L1 protein, HPV31-L1 protein, HPV33-L1 protein, HPV45-L1 protein, HPV52-L1 protein, and HPV58-L1 protein from Example 3) was used to determine its mass concentration. After serial dilution, its OD signal was detected, and the mass concentration was calculated by back-fitting the standard curve using four parameters. Each dilution experiment was repeated twice, and the average back-fitted concentration was used for fitting. Specifically, the slope of the HPV6 standard curve was 0.996, and R0... 2 The slope of the HPV11 standard curve is 0.9950; the slope of the curve is 0.9448, R 2 The slope of the HPV16 standard curve is 0.9980; the slope of the curve is 0.9599, R 2 The slope of the HPV18 standard curve is 0.9985; R is 0.9885. 2 The slope of the standard curve for HPV31 is 0.9990; the slope of the curve is 0.9942, R 2 The slope of the HPV33 standard curve is 0.9984; the slope of the curve is 0.9935, R 2 The slope of the HPV45 standard curve is 0.9988; the slope of the curve is 0.9750, R 2 The slope of the HPV52 standard curve is 0.9984; the slope of the curve is 0.9649, R 2 The slope of the HPV58 standard curve is 1.008, and the R value is 0.9987. 2 0.9990 ( Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E , Figure 3F , Figure 3G , Figure 3H , Figure 3I The linear range for HPV types 6, 11, 16, and 33 is 5-2500 ng / ml; for HPV types 18, 31, 45, and 52, it is 5-5000 ng / ml; and for HPV type 58, it is 5-10000 ng / ml, indicating that this method has good detection performance.

[0163] Example 6: Relative Accuracy

[0164] 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.

[0165] 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 2020 edition of the Chinese Pharmacopoeia and the formula under the section "I. Basic Elements of Method Validation" in the relative accuracy evaluation method. The results are shown in Table 12-20. All relative biases were within ±10%. 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).

[0166] Five concentration control groups were set up, with OD1.0 or less as the 100% potency concentration. The relative potency concentrations of other types were calculated. The average relative bias of the nine types of reagents was within ±10%, and all average relative biases met the requirements. The slope of the standard curve for HPV6 was 0.9775, for HPV11 it was 0.9455, for HPV16 it was 0.9481, for HPV18 it was 0.9985, for HPV31 it was 0.9758, for HPV33 it was 0.9733, for HPV45 it was 0.9946, for HPV52 it was 1.028, and for HPV58 it was 1.008.

[0167] Table 12 Relative Accuracy of HPV6

[0168]

[0169] Table 13 Relative Accuracy of HPV11

[0170]

[0171] Table 14 Relative Accuracy of HPV16

[0172]

[0173] Table 15 Relative Accuracy of HPV18

[0174]

[0175] Table 16 Relative Accuracy of HPV31

[0176]

[0177] Table 17 Relative Accuracy of HPV33

[0178]

[0179] Table 18 Relative Accuracy of HPV45 Model

[0180]

[0181] Table 19 Relative Accuracy of HPV52

[0182]

[0183] Table 20 Relative Accuracy of HPV58

[0184]

[0185] Example 7: Intermediate Precision

[0186] Acceptable standard: The geometrical coefficient of variation (GCV, %) of each valence level relative to the valence measurement should not exceed 20%.

[0187] 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 21-29. The geometrical coefficient of variation for each potency level's relative potency assays was less than 20%.

[0188] All nine types of reagents passed this standard in the test.

[0189] Table 21 Intermediate Precision of HPV6

[0190]

[0191] Table 22 Intermediate Precision of HPV11

[0192]

[0193] Table 23 Intermediate Precision of HPV16

[0194]

[0195] Table 24 Intermediate Precision of HPV18

[0196]

[0197] Table 25 Intermediate Precision of HPV31

[0198]

[0199] Table 26 Intermediate Precision of HPV33

[0200]

[0201] Table 27 Intermediate Precision of HPV45 Model

[0202]

[0203] Table 28 Intermediate Precision of HPV52

[0204]

[0205] Table 29 Intermediate Precision of HPV58

[0206]

Claims

1. A method for in vitro relative potency testing of an HPV vaccine or an HPV antigen bulk, characterized in that, The steps include: coating the ELISA plate with HPV non-type-specific antibodies, adding the test sample and incubating, then washing the plate; adding enzyme-labeled HPV type-specific antibodies and incubating, then washing the plate; adding chromogenic solution and developing color; adding stop solution and measuring the OD value. Further, this involves fitting a four-parameter curve Y = (da) / (1 + (X / c)^b) + a based on 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, d is the upper limit of the asymptotic range, Y is the OD value, and X is the antigen concentration. This yields the EC50 values ​​for the standard and the test sample. The in vitro relative potency of the test sample is calculated as EC50 of the test sample / EC50 of the standard. The HPV type-specific antibodies are HPV 6-specific antibodies, HPV 11-specific antibodies, HPV 16-specific antibodies, HPV 18-specific antibodies, HPV 31-specific antibodies, HPV 33-specific antibodies, HPV 45-specific antibodies, HPV 52-specific antibodies, and / or HPV 58-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 with the amino acid sequence QAS, 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 with the amino acid sequence DAS, 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 with the amino acid sequence DAS, and CDR-L3 as shown in SEQ ID NO:

24. The HPV 31-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 with the amino acid sequence RDT, and CDR-L3 as shown in SEQ ID NO:

30. The HPV 33-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 31, CDR-H2 as shown in SEQ ID NO: 32, CDR-H3 as shown in SEQ ID NO: 33, CDR-L1 as shown in SEQ ID NO: 34, CDR-L2 with the amino acid sequence DDS, and CDR-L3 as shown in SEQ ID NO:

36. The HPV 45 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 37, CDR-H2 as shown in SEQ ID NO: 38, CDR-H3 as shown in SEQ ID NO: 39, CDR-L1 as shown in SEQ ID NO: 40, CDR-L2 with the amino acid sequence GNT, and CDR-L3 as shown in SEQ ID NO:

42. The HPV 52-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 43, CDR-H2 as shown in SEQ ID NO: 44, CDR-H3 as shown in SEQ ID NO: 45, CDR-L1 as shown in SEQ ID NO: 46, CDR-L2 with the amino acid sequence GAS, and CDR-L3 as shown in SEQ ID NO:

48. The HPV 58 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 49, CDR-H2 as shown in SEQ ID NO: 50, CDR-H3 as shown in SEQ ID NO: 51, CDR-L1 as shown in SEQ ID NO: 52, CDR-L2 with the amino acid sequence DVN, and CDR-L3 as shown in SEQ ID NO:

54.

2. A method for detecting the relative potency of a 9-valent HPV vaccine or HPV antigen in liquid form, characterized in that, The steps include: adding the test sample to an ELISA plate coated with HPV non-type-specific antibodies, incubating, and washing the plate; adding enzyme-labeled HPV type-specific antibodies, incubating, and washing the plate; adding chromogenic solution and developing color; adding stop solution and measuring the OD value; 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, 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. The in vitro relative potency of the test sample = EC50 of the test sample / EC50 of the standard. The HPV type-specific antibodies include HPV 6-specific antibodies, HPV 11-specific antibodies, HPV 16-specific antibodies, HPV 18-specific antibodies, HPV 31-specific antibodies, HPV 33-specific antibodies, HPV 45-specific antibodies, HPV 52-specific antibodies, and HPV 58-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 with the amino acid sequence QAS, 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 with the amino acid sequence DAS, 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 with the amino acid sequence DAS, and CDR-L3 as shown in SEQ ID NO:

24. The HPV 31-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 with the amino acid sequence RDT, and CDR-L3 as shown in SEQ ID NO:

30. The HPV 33-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 31, CDR-H2 as shown in SEQ ID NO: 32, CDR-H3 as shown in SEQ ID NO: 33, CDR-L1 as shown in SEQ ID NO: 34, CDR-L2 with the amino acid sequence DDS, and CDR-L3 as shown in SEQ ID NO:

36. The HPV 45 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 37, CDR-H2 as shown in SEQ ID NO: 38, CDR-H3 as shown in SEQ ID NO: 39, CDR-L1 as shown in SEQ ID NO: 40, CDR-L2 with the amino acid sequence GNT, and CDR-L3 as shown in SEQ ID NO:

42. The HPV 52-specific antibody comprises CDR-H1 as shown in SEQ ID NO: 43, CDR-H2 as shown in SEQ ID NO: 44, CDR-H3 as shown in SEQ ID NO: 45, CDR-L1 as shown in SEQ ID NO: 46, CDR-L2 with the amino acid sequence GAS, and CDR-L3 as shown in SEQ ID NO:

48. The HPV 58 specific antibody comprises CDR-H1 as shown in SEQ ID NO: 49, CDR-H2 as shown in SEQ ID NO: 50, CDR-H3 as shown in SEQ ID NO: 51, CDR-L1 as shown in SEQ ID NO: 52, CDR-L2 with the amino acid sequence DVN, and CDR-L3 as shown in SEQ ID NO:

54.

3. The method according to claim 1, wherein the ELISA plate is pre-made, and the sample to be tested is added to the pre-made ELISA plate coated with HPV non-type-specific antibodies for incubation, and the detection method does not include the step of using HPV non-type-specific antibodies to coat the plate.

4. The detection method according to claim 1 or claim 2, wherein, The HPV 6-specific antibody comprises VH as shown in SEQ ID NO: 55 and VL as shown in SEQ ID NO: 56; The HPV 11 specific antibody comprises VH as shown in SEQ ID NO: 57 and VL as shown in SEQ ID NO: 58; The HPV 16 specific antibody comprises VH as shown in SEQ ID NO: 59 and VL as shown in SEQ ID NO: 60; The HPV 18 specific antibody comprises VH as shown in SEQ ID NO: 61 and VL as shown in SEQ ID NO: 62; The HPV 31 specific antibody comprises VH as shown in SEQ ID NO: 63 and VL as shown in SEQ ID NO: 64; The HPV 33-specific antibody comprises VH as shown in SEQ ID NO: 65 and VL as shown in SEQ ID NO: 66; The HPV 45 specific antibody comprises VH as shown in SEQ ID NO: 67 and VL as shown in SEQ ID NO: 68; The HPV 52-specific antibody comprises VH as shown in SEQ ID NO: 69 and VL as shown in SEQ ID NO: 70; The HPV 58 specific antibody comprises VH as shown in SEQ ID NO: 71 and VL as shown in SEQ ID NO:

72.

5. The detection method according to claim 1 or 2, wherein the HPV type-specific antibody comprises, for example: (1) the heavy chain constant region shown in SEQ ID NO: 93, and (2) the light chain constant region shown in either SEQ ID NO: 91 or SEQ ID NO:

92.

6. The detection method according to claim 1 or 2, 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.

7. The detection method according to claim 6, wherein the enzyme-labeled HPV type-specific antibody is a horseradish peroxidase-labeled HPV type-specific antibody.

8. The method according to claim 1 or 2, specifically comprising: (1) Equilibrate all reagents to room temperature; (2) Prepare the solution by diluting the washing solution with distilled or deionized water to the concentration to be used; (3) Sample processing: Pre-dilution of the standard and the sample to be tested. The pre-dilution concentration of HPV6 is 5000-20000 ng / mL, the pre-dilution concentration of HPV11 is 10000-20000 ng / mL, the pre-dilution concentration of HPV16 is 2500-10000 ng / mL, the pre-dilution concentration of HPV18 is 5000-20000 ng / mL, the pre-dilution concentration of HPV31 is 5000-20000 ng / mL, the pre-dilution concentration of HPV33 is 5000-20000 ng / mL, the pre-dilution concentration of HPV45 is 5000-20000 ng / mL, the pre-dilution concentration of HPV52 is 5000-20000 ng / mL, and the pre-dilution concentration of HPV58 is 5000-20000 ng / mL. Then, serially dilute by 2 times, for a total of 6-11 dilutions. (4) Add the diluted sample and positive and negative controls to the corresponding wells of the ELISA plate; (5) Incubation: Incubate at 37°C in a constant temperature incubator; (6) Washing: After incubation, 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 dilution buffer, and add the diluted detection antibody to each well; (8) Incubation: Incubate at 37°C in a constant temperature incubator; (9) After washing and incubation, carefully remove the sealing film, discard the liquid in the wells, add diluted washing solution, and wash the plate. (10) For 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. (11) Termination / reading: Remove the sealing film, add the termination solution to each well, mix well, 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: Fit a four-parameter curve Y = (da) / (1 + (X / c)^b) + a based on 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, d is the upper limit of the asymptote, Y is the OD value, and X is the antigen concentration.

9. The method according to claim 1 or 2, wherein the standard is as follows: 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 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%. 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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