CN121299112B - A method for detecting the relative efficacy of HPV type 9 vaccines or antigens in liquid form - Google Patents
A method for detecting the relative efficacy of HPV type 9 vaccines or antigens in liquid formInfo
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Abstract
本发明涉及一种检测九型HPV疫苗或抗原原液体外相对效力的方法，具体应用酶联免疫法检测HPV‑VLP抗原原液和疫苗成品(6、11、16、18、31、33、45、52和58型)的体外相对效力。本发明的酶联免疫法具有优良的准确度、精密度，提升了HPV疫苗质量评价的标准化水平，预期对推动高质量HPV疫苗的研发奠定基础。
This invention relates to a method for detecting the relative in vitro potency of HPV type 9 vaccines or antigen stock solutions. Specifically, it applies enzyme-linked immunosorbent assay (ELISA) to detect the relative in vitro potency of HPV-VLP antigen stock solutions and finished vaccine products (types 6, 11, 16, 18, 31, 33, 45, 52, and 58). The ELISA method of this invention has excellent accuracy and precision, improving the standardization level of HPV vaccine quality evaluation and is expected to lay the foundation for promoting the research and development of high-quality HPV vaccines.
Description
Technical Field
The invention relates to the field of detection, in particular to an enzyme-linked immunosorbent assay for detecting the in vitro relative efficacy of HPV vaccine or antigen stock solution, in particular to the detection of nine-valent (6, 11, 16, 18, 31, 33, 45, 52, 58) HPV vaccine.
Background
Human papilloma virus (human papillomavirus, HPV) infects humans extensively, and can cause the occurrence of malignant tumors such as cervical cancer, which seriously jeopardizes human health. HPV vaccines are effective against HPV infection and the diseases caused by HPV infection, and HPV vaccines which have entered clinical research contain polytype antigens (2, 3,4, 9, 11, 14, 15).
The in vitro efficacy experiment with good correlation with in vivo efficacy can replace the in vivo efficacy experiment of mice to be used for release detection of vaccines, so that the time is saved and the use of animals is reduced. In vitro relative potency is a critical quality attribute of HPV vaccines, quality control requires the determination of type-specific antigen content. At present, each production enterprise utilizes respective HPV type specific monoclonal antibodies to establish a double-antibody sandwich ELISA method to detect respective L1 antigens, but the detection results are large in difference and inconsistent in standard, and partial research and development units lack dominant monoclonal antibodies for recognizing key specific epitopes, so that HPV vaccine research and development and detection standardization are limited. The HPV type specific monoclonal antibody needs to be developed, a unified and standardized HPV type specific antigen quantification method is established, the standardization level of HPV vaccine quality evaluation is improved, and the development of high-quality HPV vaccine is promoted.
Disclosure of Invention
On one hand, the invention provides a method for detecting the relative efficacy of HPV vaccine or HPV antigen stock solution outside the liquid, which is characterized by comprising the following steps of coating an ELISA plate by using an HPV non-type specific antibody, adding a sample to be detected for incubation, washing the plate, adding an enzyme-labeled HPV type specific antibody for incubation, washing the plate, adding a color development liquid for color development, adding a stop solution, and measuring an OD value. In some embodiments, the method further comprises detecting a standard, the method of detecting the standard being identical to the sample to be tested. In some embodiments, the method further comprises fitting a four parameter curve y= (d-a)/(1+ (X/c)/(b) +a, where a is the lower asymptote limit, b is the linear interval straight line slope, c is the curve between 50% reaction points (a and d) (expressed as concentration), d is the upper asymptote limit, Y is the OD value, X is the antigen concentration, and EC50 values for the standard and the test sample are obtained, and the in vitro relative efficacy of the test sample = EC50 of the test sample/EC 50 of the standard. In some embodiments, the elisa plate is prefabricated, the sample to be tested is added to the HPV non-type specific antibody coated prefabricated elisa plate for incubation, and the detection method does not include the step of using HPV non-type specific antibody coated plates.
On the other hand, the invention also provides a method for detecting the external relative efficacy of a 9-valent HPV vaccine or HPV antigen stock solution, which is characterized by comprising the steps of adding a sample to be detected to an ELISA plate coated with HPV non-type specific antibodies, incubating, washing the plate, adding enzyme-labeled HPV type specific antibodies, incubating, washing the plate, adding a color development solution, developing, adding a stop solution, measuring an OD value, fitting a four-parameter curve Y= (d-a)/(1+ (X/c)/(b) +a according to the antigen concentration and the OD value, wherein a is the lower limit of an asymptote, b is the linear interval linear slope, c is the reaction point (between a and d) (expressed as concentration) of a curve 50%, d is the upper limit of the asymptote, Y is the OD value, X is the antigen concentration, and the EC50 values of a standard sample and the sample to be detected are obtained, and the external relative efficacy of the sample to be detected = the EC50 of the sample to be detected/the EC50 of the standard sample;
The HPV type-specific antibodies comprise 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.
In another aspect, the invention also provides an ELISA kit comprising an ELISA plate coated with an HPV non-type specific antibody. In some embodiments, the kit is for in vitro relative potency detection of HPV vaccine or HPV antigen stock, the kit further comprising a detection antibody that is an enzyme-labeled HPV type-specific antibody, wherein the HPV type-specific antibody is selected from the group consisting of 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.
In some embodiments, the HPV antigen stock solution is an HPV type 6, the HPV type-specific antibody is an HPV antibody that is HPV 6-specific, 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.
In some embodiments, the HPV antigen stock is HPV type 11, 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.
In some embodiments, the HPV antigen stock solution is HPV type 16, the HPV type-specific antibody is an HPV antibody specific for HPV16, 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.
In some embodiments, the HPV antigen stock is HPV type 18, the HPV type-specific antibody is an HPV antibody specific for HPV18, 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.
In some embodiments, the HPV antigen stock solution is HPV type 31 and the HPV type-specific antibody is an HPV antibody specific for HPV31 comprising CDR-H1 as shown in SEQ ID NO:25, CDR-H2 as shown in SEQ ID NO:26, CDR-H3 as shown in SEQ ID NO:27, CDR-L1 as shown in SEQ ID NO:28, CDR-L2 as shown in SEQ ID NO:29 and CDR-L3 as shown in SEQ ID NO: 30.
In some embodiments, the HPV antigen stock solution is HPV33 type, the HPV type-specific antibody is an HPV 33-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.
In some embodiments, the HPV antigen stock solution is HPV45 type, the HPV type-specific antibody is an HPV 45-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.
In some embodiments, the HPV antigen stock solution is HPV type 52, the HPV type-specific antibody is an HPV antibody specific for HPV52, 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.
In some embodiments, the HPV antigen stock solution is HPV type 58, the HPV type-specific antibody is an HPV antibody specific for HPV58, 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.
In some embodiments, the HPV vaccine is an HPV 9-valent vaccine, and 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 HPV 58-specific antibodies:
The HPV 6 specific antibody comprises CDR-H1 shown as SEQ ID NO. 1, CDR-H2 shown as SEQ ID NO. 2, CDR-H3 shown as SEQ ID NO. 3, CDR-L1 shown as SEQ ID NO. 4, CDR-L2 shown as SEQ ID NO. 5 and CDR-L3 shown as SEQ ID NO. 6;
The HPV 11 specific antibody comprises CDR-H1 shown as SEQ ID NO. 7, CDR-H2 shown as SEQ ID NO. 8, CDR-H3 shown as SEQ ID NO. 9, CDR-L1 shown as SEQ ID NO. 10, CDR-L2 shown as SEQ ID NO. 11 and CDR-L3 shown as SEQ ID NO. 12;
the HPV 16 specific antibody comprises CDR-H1 shown as SEQ ID NO. 13, CDR-H2 shown as SEQ ID NO. 14, CDR-H3 shown as SEQ ID NO. 15, CDR-L1 shown as SEQ ID NO. 16, CDR-L2 shown as SEQ ID NO. 17 and CDR-L3 shown as SEQ ID NO. 18;
the HPV 18 specific antibody comprises CDR-H1 shown as SEQ ID NO. 19, CDR-H2 shown as SEQ ID NO. 20, CDR-H3 shown as SEQ ID NO. 21, CDR-L1 shown as SEQ ID NO. 22, CDR-L2 shown as SEQ ID NO. 23 and CDR-L3 shown as SEQ ID NO. 24;
the HPV 31 specific antibody comprises CDR-H1 shown in SEQ ID NO. 25, CDR-H2 shown in SEQ ID NO. 26, CDR-H3 shown in SEQ ID NO. 27, CDR-L1 shown in SEQ ID NO. 28, CDR-L2 shown in SEQ ID NO. 29 and CDR-L3 shown in SEQ ID NO. 30;
the HPV 33 specific antibody comprises CDR-H1 shown in SEQ ID NO. 31, CDR-H2 shown in SEQ ID NO. 32, CDR-H3 shown in SEQ ID NO. 33, CDR-L1 shown in SEQ ID NO. 34, CDR-L2 shown in SEQ ID NO. 35 and CDR-L3 shown in SEQ ID NO. 36;
the HPV 45 specific antibody comprises CDR-H1 shown in SEQ ID NO. 37, CDR-H2 shown in SEQ ID NO. 38, CDR-H3 shown in SEQ ID NO. 39, CDR-L1 shown in SEQ ID NO. 40, CDR-L2 shown in SEQ ID NO. 41 and CDR-L3 shown in SEQ ID NO. 42;
The HPV 52 specific antibody comprises CDR-H1 shown as SEQ ID NO. 43, CDR-H2 shown as SEQ ID NO. 44, CDR-H3 shown as SEQ ID NO. 45, CDR-L1 shown as SEQ ID NO. 46, CDR-L2 shown as SEQ ID NO. 47 and CDR-L3 shown as SEQ ID NO. 48;
The HPV 58 specific antibody comprises CDR-H1 shown in SEQ ID NO. 49, CDR-H2 shown in SEQ ID NO. 50, CDR-H3 shown in SEQ ID NO. 51, CDR-L1 shown in SEQ ID NO. 52, CDR-L2 shown in SEQ ID NO. 53 and CDR-L3 shown in SEQ ID NO. 54.
In some embodiments, the acceptable criteria for the method are as follows:
① In the four-parameter fitting curve, the regression determination coefficient R 2 is more than or equal to 0.98, the d-a of the four-parameter curve is more than or equal to 2.0, and the ratio of the b value of the sample curve to the reference curve is between 0.8 and 1.2;
② The 10% -75% value of the upper limit and the lower limit of the OD is defined as a linear interval, namely OD10% = a+10% × (d-a), OD75% = a+75% × (d-a), the number of linear effective concentration points in the dilution gradient range of each sample is required to be more than or equal to 3, and CV (constant velocity) among multiple holes in each linear interval of the sample is less than or equal to 30%;
③ The OD value of the negative control hole is less than or equal to 0.1, the OD value of the positive control hole is more than or equal to 0.8.
If one of the above criteria is not met, the test is not effective.
In some embodiments, the HPV type-specific antibody is selected from the group consisting of an HPV 6-specific antibody, an HPV 11-specific antibody, an HPV 16-specific antibody, an HPV 18-specific antibody, an HPV 31-specific antibody, an HPV 33-specific antibody, an HPV 45-specific antibody, an HPV 52-specific antibody and/or an HPV 58-specific antibody, wherein,
The HPV 6 specific antibody comprises CDR-H1 shown as SEQ ID NO. 1, CDR-H2 shown as SEQ ID NO. 2, CDR-H3 shown as SEQ ID NO. 3, CDR-L1 shown as SEQ ID NO. 4, CDR-L2 shown as SEQ ID NO. 5 and CDR-L3 shown as SEQ ID NO. 6;
The HPV 11 specific antibody comprises CDR-H1 shown as SEQ ID NO. 7, CDR-H2 shown as SEQ ID NO. 8, CDR-H3 shown as SEQ ID NO. 9, CDR-L1 shown as SEQ ID NO. 10, CDR-L2 shown as SEQ ID NO. 11 and CDR-L3 shown as SEQ ID NO. 12;
the HPV 16 specific antibody comprises CDR-H1 shown as SEQ ID NO. 13, CDR-H2 shown as SEQ ID NO. 14, CDR-H3 shown as SEQ ID NO. 15, CDR-L1 shown as SEQ ID NO. 16, CDR-L2 shown as SEQ ID NO. 17 and CDR-L3 shown as SEQ ID NO. 18;
the HPV 18 specific antibody comprises CDR-H1 shown as SEQ ID NO. 19, CDR-H2 shown as SEQ ID NO. 20, CDR-H3 shown as SEQ ID NO. 21, CDR-L1 shown as SEQ ID NO. 22, CDR-L2 shown as SEQ ID NO. 23 and CDR-L3 shown as SEQ ID NO. 24;
the HPV 31 specific antibody comprises CDR-H1 shown in SEQ ID NO. 25, CDR-H2 shown in SEQ ID NO. 26, CDR-H3 shown in SEQ ID NO. 27, CDR-L1 shown in SEQ ID NO. 28, CDR-L2 shown in SEQ ID NO. 29 and CDR-L3 shown in SEQ ID NO. 30;
the HPV 33 specific antibody comprises CDR-H1 shown in SEQ ID NO. 31, CDR-H2 shown in SEQ ID NO. 32, CDR-H3 shown in SEQ ID NO. 33, CDR-L1 shown in SEQ ID NO. 34, CDR-L2 shown in SEQ ID NO. 35 and CDR-L3 shown in SEQ ID NO. 36;
the HPV 45 specific antibody comprises CDR-H1 shown in SEQ ID NO. 37, CDR-H2 shown in SEQ ID NO. 38, CDR-H3 shown in SEQ ID NO. 39, CDR-L1 shown in SEQ ID NO. 40, CDR-L2 shown in SEQ ID NO. 41 and CDR-L3 shown in SEQ ID NO. 42;
The HPV 52 specific antibody comprises CDR-H1 shown as SEQ ID NO. 43, CDR-H2 shown as SEQ ID NO. 44, CDR-H3 shown as SEQ ID NO. 45, CDR-L1 shown as SEQ ID NO. 46, CDR-L2 shown as SEQ ID NO. 47 and CDR-L3 shown as SEQ ID NO. 48;
The HPV 58 specific antibody comprises CDR-H1 shown in SEQ ID NO. 49, CDR-H2 shown in SEQ ID NO. 50, CDR-H3 shown in SEQ ID NO. 51, CDR-L1 shown in SEQ ID NO. 52, CDR-L2 shown in SEQ ID NO. 53 and CDR-L3 shown in SEQ ID NO. 54.
In some embodiments, the HPV 6-specific antibody comprises a VH as shown in SEQ ID NO:55 and a VL as shown in SEQ ID NO: 56.
In some embodiments, the HPV 11-specific antibody comprises a VH as shown in SEQ ID NO:57 and a VL as shown in SEQ ID NO: 58.
In some embodiments, the HPV 16-specific antibody comprises a VH as shown in SEQ ID NO:59 and a VL as shown in SEQ ID NO: 60.
In some embodiments, the HPV 18 specific antibody comprises a VH as shown in SEQ ID NO:61 and a VL as shown in SEQ ID NO: 62.
In some embodiments, the HPV 31-specific antibody comprises a VH as shown in SEQ ID NO:63 and a VL as shown in SEQ ID NO: 64.
In some embodiments, the HPV 33 specific antibody comprises a VH as shown in SEQ ID NO:65 and a VL as shown in SEQ ID NO: 66.
In some embodiments, the HPV 45-specific antibody comprises a VH as shown in SEQ ID NO:67 and a VL as shown in SEQ ID NO: 68.
In some embodiments, the HPV 52 specific antibody comprises a VH as shown in SEQ ID NO:69 and a VL as shown in SEQ ID NO: 70.
In some embodiments, the HPV 58 specific antibody comprises a VH as shown in SEQ ID NO:71 and a VL as shown in SEQ ID NO: 72.
In some embodiments, the HPV type-specific antibody comprises a heavy chain constant region as shown in (1) SEQ ID NO. 93 and (2) a light chain constant region as shown in any one of SEQ ID NO. 91 and SEQ ID NO. 92.
A polynucleotide encoding a type-specific antibody of the invention. In some embodiments, the polynucleotide is selected from the group consisting of (1) SEQ ID NOS: 73 and 74, (2) SEQ ID NOS: 75 and 76, (3) SEQ ID NOS: 77 and 78, (4) SEQ ID NOS: 79 and 80, (5) SEQ ID NOS: 81 and 82, (6) SEQ ID NOS: 83 and 84, (7) SEQ ID NOS: 85 and 86, (8) SEQ ID NOS: 87 and 88, and (9) SEQ ID NOS: 89 and 90.
In some embodiments, the method is an HPV vaccine in vitro relative potency detection method, further comprising the step of desorbing.
In some embodiments, the detection antibody is an enzyme-labeled detection antibody. In some embodiments, the enzyme-labeled detection antibody is horseradish peroxidase (Horseradish Peroxidase, HRP), alkaline phosphatase (AlkalinePhosphatase, AKP), beta-galactosidase (beta-Galactosidase), glucose Oxidase (GOD), acid phosphatase-labeled detection antibody. 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.
In some embodiments, the kit further comprises a sample diluent, an enzyme-labeled antibody diluent, a chromogenic solution, a stop solution, and a wash solution.
In some embodiments, the detection method specifically comprises:
(1) Equilibration, equilibrate all reagents to room temperature;
(2) Preparing a washing solution, namely diluting the washing solution to the use concentration by distilled water or deionized water;
(3) Sample treatment, namely pre-diluting a standard substance and a sample to be tested, wherein the concentration of Sa01 after HPV6 pre-dilution is 5000-20000ng/mL, the concentration of Sa01 after HPV11 pre-dilution is 10000-20000ng/mL, the concentration of Sa01 after HPV16 pre-dilution is 2500-10000ng/mL, the concentration of Sa01 after HPV18 pre-dilution is 5000-20000ng/mL, the concentration of Sa01 after HPV31 pre-dilution is 5000-20000ng/mL, the concentration of Sa01 after HPV33 pre-dilution is 5000-20000ng/mL, the concentration of Sa01 after HPV45 pre-dilution is 5000-20000ng/mL, the concentration of Sa01 after HPV52 pre-dilution is 5000-20000ng/mL (the marked concentration of the sample to be tested is regarded as initial concentration, dilution is the target concentration) and then serial dilution of 2 times or 3 times, and the total dilution is 6-11 degrees;
(4) Adding a sample, namely adding diluted samples and negative and positive controls into corresponding holes of the ELISA plate;
(5) Incubation, wherein incubation is carried out in a 37 ℃ constant temperature incubator;
(6) After washing and incubation, carefully uncovering the sealing plate film, discarding the liquid in the hole, adding diluted washing liquid, and washing the plate;
(7) Adding enzyme, diluting the detection antibody to the use concentration by using enzyme-labeled antibody diluent, and adding diluted detection antibody into each hole;
(8) Incubation, wherein incubation is carried out in a 37 ℃ constant temperature incubator;
(9) After washing and incubation, carefully uncovering the sealing plate film, discarding the liquid in the hole, adding diluted washing liquid, and washing the plate;
(10) Color development, adding color development liquid into each hole, and incubating in a 37 ℃ constant temperature incubator in a dark place after sealing by a sealing plate membrane;
(11) Stopping/reading, removing the sealing plate film, adding a stopping solution to each well, mixing, reading (set to 620nm wavelength, and subtracting the reading at 620nm from the reading at 450nm to correct the optical defect in the plate), and (12) data processing, fitting a four parameter curve Y= (d-a)/(1+ (X/c)/(b) +a according to the antigen concentration and OD value, wherein a is the lower asymptote limit, b is the linear interval straight line slope, c is the curve 50% between the reaction points (a and d) (expressed as concentration), d is the upper asymptote limit, Y is the OD value, and X is the antigen concentration.
In some embodiments, the assay method further comprises (13) calculating in vitro relative potency, in vitro relative potency = EC50 of test sample/EC 50 of standard.
Those skilled in the art will appreciate that HPV type-specific antibodies correspond to the type of vaccine or antigen stock to be tested. For example, when the vaccine or antigen stock solution to be tested is HPV 6 type, the HPV type specific antibody is HPV 6 specific antibody, when the vaccine or antigen stock solution to be tested is HPV11 type, the HPV type specific antibody is HPV11 specific antibody, when the vaccine to be tested is HPV 6 or HPV162 type, the HPV type specific antibody is HPV 6 specific antibody and HPV16 specific antibody, when the vaccine to be tested is HPV 6, HPV11, HPV16 or HPV184 type, the HPV type specific antibody is HPV 6 specific antibody, HPV11 specific antibody, HPV16 specific antibody and HPV18 specific antibody, when the vaccine to be tested is HPV 6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV589 type specific antibody is HPV 6 specific antibody, HPV11 specific antibody, HPV16 specific antibody, HPV18 specific antibody, HPV31 specific antibody, HPV33 specific antibody, HPV45 specific antibody, HPV52 specific antibody and HPV58 specific antibody.
It will be appreciated by those skilled in the art that when the sample to be tested is a multivalent vaccine (e.g., 9-valent), detection is performed using detection antibodies corresponding to the HPV antigen types contained therein, respectively. For example, when the sample to be tested is HPV 6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV589 vaccine, detection antibodies specific for HPV 6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58 types are used for detection, respectively. The ELISA detection kit of the invention can be a kit for detecting single HPV antigens, such as a kit for detecting HPV 6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58, or a kit for detecting multivalent vaccines, such as a kit for detecting HPV 6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV589 vaccine, and the kit contains 9 detection antibodies.
The method has high accuracy, good precision and high specificity, improves the standardization level of HPV vaccine quality evaluation, and lays a foundation for promoting the research and development of high-quality HPV vaccine.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the layout of an ELISA plate used in the detection method of the invention;
FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 2I show the results of HPV6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 45, HPV 52, HPV 58 antigen stock solution detection, respectively, for manufacturer 1;
fig. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I show linear regression equations for HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 45, HPV 52, HPV 58 nine VLP theoretical log values and potency theoretical log values, respectively;
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.
In the present invention, peripheral blood was collected from 14 adult volunteers after completing HPV nine-valent vaccination for one month, and upper plasma and middle PBMCs were obtained by density gradient centrifugation. The fluorescent marked HPV6, 11, 16, 18, 31, 33, 45, 52 and 58 type L1 proteins are used for separating the memory B cells which are specifically combined with the 9 types of the proteins from PBMC through flow sorting, a transfectable PCR fragment with the expression activity is obtained through nest PCR, the transfected CHO cells are used for expression, cell supernatant containing secretion antibodies is obtained, the binding activity screening is carried out through ELISA, 2475 types of positive binding clones are obtained, 90 types of clones are specifically combined with HPV6 type L1 proteins, 181 types of clones are specifically combined with HPV11 type L1 proteins, 130 types of clones are specifically combined with HPV16 type L1 proteins, 97 types of clones are specifically combined with HPV18 type L1 proteins, 94 types of clones are specifically combined with HPV31 type L1 proteins, 87 types of clones are specifically combined with HPV33 type L1 proteins, 88 types of clones are specifically combined with HPV45 type L1 proteins, 90 types of clones are specifically combined with HPV52 type L1 proteins, 79 types of clones are specifically combined with HPV58 type L1 proteins, and at least two types of clones are specifically combined with HPV1 type L1. Based on the binding of cell supernatant and the neutralization result of pseudovirus, the above 10 kinds of antibodies were constructed as 25, 20, 35, 44, 42, 37, 26, 40, 23 and 63 recombinant antibodies, respectively. Combining the binding activity of each recombinant antibody with the corresponding type protein and the neutralization activity of each pseudovirus, and finally selecting the anti-HPV 6 antibody F5-222, the anti-HPV 11 antibody F5-422, the anti-HPV 16 antibody F5-194, the anti-HPV 18 antibody F5-212, the anti-HPV 31 antibody F5-183, the anti-HPV 33 antibody F5-155, the anti-HPV 45 antibody F5-160, the anti-HPV 52 antibody F5-398 and the anti-HPV 58 antibody F5-354 respectively by 10 types of antibodies. The specific antibodies of the type all specifically bind and neutralize the proteins or pseudoviruses of the type, the binding activity EC50 is lower than 40ng/mL, and the neutralizing activity IC50 is between 0.25 and 51.60 ng/mL.
TABLE 1 description of amino acid sequences and nucleic acid sequences
Example 1 preparation of anti-HPV antibodies
Cloning the coding sequences of the light and heavy chain variable regions of the antibody into eukaryotic expression vectors carrying coding sequences of human IgG1 constant regions, transiently transfecting CHO cells, performing secretory expression, and obtaining 9 anti-HPV antibody clones with purity of >90% by affinity purification, namely F5-222, F5-422, F5-194, F5-212, F5-183, F5-155, F5-160, F5-398 and F5-354.
The amino acid sequences of VH, VL and CL of the antibodies are shown in table 1.
Amino acid sequence of human IgG1 constant region:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Example 2 characterization of anti-HPV antibodies-characterization of antigen binding specificity by ELISA
HPV 9 type L1 protein [ HPV6 (genbank: UNG 35082.1), HPV11 (genbank: AAA 46935.1), HPV16 (genbank: QGC 89586.1), HPV18 (genbank: ACU 01871.1), HPV31 (genbank: OP 900721.1), HPV33 (genbank: WAN 40740.1), HPV45 (genbank: AAY 86494.1), HPV52 (genbank: BBD 06702.1) and HPV58 (genbank: WAN 40708.1) ] diluted to 2. Mu.g/mL with PBS, respectively, was added to 96-well ELISA plates (NEST, 504201) at 100. Mu.L/well and coated overnight at 4 ℃. The solution was removed, washed 2 times with PBST and blocked with blocking solution (pbs+5%bsa) for 2 hours at 37 ℃. The solution was removed and antibody (concentration 1. Mu.g/mL) diluted with diluent (PBS+5% BSA) was added to the microplate at 100. Mu.L/well and incubated at 37℃for 1 hour. The solution was removed, washed 3 times with PBST, 100. Mu.L of 1W-fold diluted mouse anti-human IgG Fc-HRP (Vazyme self-produced) was added to each well and incubated at 37℃for 1 hour. The solution was removed and washed 3 times with PBST, 100. Mu.L of chromogenic substrate TMB was added to each well and incubated at 37℃for 10 minutes in the absence of light. The solution was removed and washed 3 times with PBST and 50. Mu.L of 2M sulfuric acid was added to each well. OD at 450nm was measured on a multifunctional microplate reader (Tecan, spark). The results are shown in Table 2.
TABLE 2 protein binding Activity of anti-HPV antibodies
The results show that F5-222, F5-422, F5-194, F5-212, F5-183, F5-155, F5-160, F5-398, F5-354 bind only to a single type of protein, with specificity.
EXAMPLE 3 ELISA method for detecting relative potency of nine-valent (6, 11, 16, 18, 31, 33, 45, 52, 58) human papillomavirus vaccine in vitro
Material preparation
The ELISA plate is prepared by taking three non-type specific antibodies prepared in the embodiment 1, adding the three antibodies in equal proportion by using coating liquid (0.05M carbonate buffer), namely, adding 100 mu L of the mixture into each hole of a 96-hole ELISA plate, and placing the mixture at 2-8 ℃ for adsorption for 24 hours. Removing the coating liquid, and washing the plate 3 times by using coating washing liquid (0.2 mol/LPBST);
Enzyme-labeled reagent, wherein the enzyme-labeled reagent comprises horseradish peroxidase labeled F5-222, F5-422, F5-194, F5-212, F5-183, F5-155, F5-160, F5-398 and F5-354 with the concentration of 5 mg/ml;
Enzyme-labeled reagent diluent, 0.1mol/L PBS,0.05% Tween 20,1% BSA,0.1% P300 preservative
Color development liquid TMB color development liquid (Yingchang biotechnology, product number EL 0001)
Stopping solution, namely adding 1M sulfuric acid with ultrapure water to prepare a solution with the concentration of 2mol/L
Sample dilution 0.1mol/LPBS,0.05% Tween 20,0.5% Casein,0.1% P300 preservative
The concentrated washing solution is PBST, 0.2mol/L, PBS with pH of 7.4 and Tween 20 with volume ratio of 0.05% are added
Positive control diluent 0.1mol/L PBS,0.05% Tween 20,0.5% Casein,0.1% P300 preservative
Negative control, 0.1mol/L PBS,0.05% Tween 20,0.5% Casein,0.1% P300 preservative
Standard substances are 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 (manufacturer 1 self-made, E.coli)
Samples to be tested were antigen stock from manufacturer 1 (3 batches each of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58 antigen stock).
Experimental method
1. Equilibration-all reagents were equilibrated to room temperature (at least 30 min) and frozen samples were mixed.
2. Preparing a concentrated washing solution, namely diluting the concentrated washing solution with distilled water by 20 times.
3. Sample treatment, namely pre-diluting a standard substance and a sample to be tested, wherein the concentration of Sa01 after HPV6 pre-dilution is 5000-10000ng/mL, the concentration of Sa01 after HPV11 pre-dilution is 10000-20000ng/mL, the concentration of Sa01 after HPV16 pre-dilution is 2500-10000ng/mL, the concentration of Sa01 after HPV18 pre-dilution is 5000-20000ng/mL, the concentration of Sa01 after HPV31 pre-dilution is 10000-20000ng/mL, the concentration of Sa01 after HPV33 pre-dilution is 10000-20000ng/mL, the concentration of Sa01 after HPV45 pre-dilution is 1000-20000ng/mL, the concentration of Sa01 after HPV52 pre-dilution is 1000-10000ng/mL, the concentration of Sa01 after HPV58 pre-dilution is 1000-10000ng/mL (the marked concentration of the sample to be tested is regarded as initial concentration, and diluted to the target concentration), and then serial dilution is performed by 2 times, and 11 dilutions are performed for 2 multiple wells.
4. And (3) adding samples, namely arranging plates (corresponding to each type of enzyme label plate) according to the figure 1, adding 100 mu L of diluted standard substance or diluted corresponding sample to be tested into corresponding holes, and carrying out yin-yang comparison on the corresponding holes by 4 holes. For example, HPV type 6 standard concentration gradient after dilution is 9.8-10000ng/mL, and the plate layout is shown in FIG. 1.
5. Incubation, namely placing the membrane sealing plate into a 37 ℃ constant temperature incubator for incubation for 30min.
6. After the incubation is completed, the sealing plate film is carefully removed, the liquid in the holes is discarded, at least 300 mu L of 1 Xwashing liquid is added into each hole, the washing liquid is discarded after standing for 30 seconds, the plate is continuously washed for 5 times, and the residual liquid is removed as much as possible in the last time.
7. Enzyme-labeled reagent (100X) was diluted to 1X with enzyme-labeled reagent dilution, and 100. Mu.L of enzyme-labeled reagent was added to each well.
8. Incubation, namely placing the membrane sealing plate into a 37 ℃ constant temperature incubator for incubation for 30min.
9. And (6) repeating the step 6.
10. And (3) color development, namely adding 100 mu L of color development liquid into each hole, sealing the holes by using a sealing plate membrane, and then placing the holes into a 37 ℃ constant temperature incubator for light-proof incubation for 15min.
11. Stop/read value the sealing plate film was carefully removed, 50. Mu.L of stop solution was added to each well and gently mixed to obtain the read value. Set to a wavelength of 620nm and subtract the reading at 620nm from the reading at 450nm (correct for optical defects in the plate).
12. And (3) data processing, namely performing four-parameter curve fitting by using Soft Max 4.8 edition (PERKIN ELMER multifunctional enzyme-labeled instrument self-contained software). The difference between the EC50 of the sample to be tested and the EC50 of the standard is analyzed, and the in vitro relative efficacy of the sample to be tested is evaluated, wherein the in vitro relative efficacy=ec 50 of the sample to be tested/EC 50 of the standard, and the results are shown in fig. 2A, fig. 2B, fig. 2C, fig. 2D, fig. 2E, fig. 2F, fig. 2G, fig. 2H, and fig. 2I.
Four-parameter curve fitting equation Y= (d-a)/(1+ (X/c)/(b) +a)
A=asymptote lower limit
B=linear interval straight line slope
C=curve 50% reaction point (between a and d) (expressed as concentration)
D=asymptote upper limit
Y=od value
X = concentration
13. Experimental acceptable standards:
① In the four-parameter fitting curve, the regression determination coefficient R 2 is more than or equal to 0.98, the d-a of the four-parameter curve is more than or equal to 2.0, and the ratio of the b value of the sample curve to the reference curve is between 0.8 and 1.2;
② The 10% -75% value of the upper limit and the lower limit of the OD is defined as a linear interval, namely OD10% = a+10% × (d-a), OD75% = a+75% × (d-a), the number of linear effective concentration points in the dilution gradient range of each sample is required to be more than or equal to 3, and CV (constant velocity) among multiple holes in each linear interval of the sample is less than or equal to 30%;
③ The OD value of the negative control hole is less than or equal to 0.1, the OD value of the positive control hole is more than or equal to 0.8.
EXAMPLE 4 specificity
The method is used for researching the fact that HPV antigens of different types are added into HPV single type antigens to be detected, namely, the capability of mixing nine HPV type antigens and detecting the titers of the other eight antigens by the type specific detection antibodies is examined. The specificity evaluation was performed by calculating the test concentration/theoretical concentration ratio (%), i.e., the relative bias (%). Nine reagent relative bias averages met this criterion, with 30% as the standard.
The assay was performed using the standard and antigen stock of manufacturer 1 of example 3 in substantially the same manner as in example 3 (except that when each type of antigen was added to the sample to be tested, an equal proportion of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58 antigen stock was added).
The specific results of HPV nine-type reagent detection are shown in tables 3-11.
TABLE 3 HPV6 specificity
TABLE 4 HPV11 specificity
TABLE 5 HPV16 specificity
TABLE 6 HPV18 specificity
TABLE 7 HPV31 specificity
TABLE 8 HPV33 specificity
TABLE 9 HPV45 specificity
TABLE 10 HPV52 specificity
TABLE 11 HPV58 specificity
Example 5 Standard Curve and Linear
Acceptable criteria are that the linear regression is performed by the least squares method by plotting the logarithm of theoretical concentration values (abscissa) against the logarithm of concentration measurements (ordinate). The correlation coefficient of the linear regression equation should be not lower than 0.98. For relative accuracy, intermediate precision and potency are consistent with concentration levels at levels, which range should cover at least 80% -150% of the concentration levels.
As a result of the verification, since no HPV-VLP standard was used, the mass concentration was determined using vaccine manufacturer 1 antigen (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 of example 3), OD signals were detected after gradient dilution, and the mass concentration was calculated by four-parameter fitting. Each dilution experiment was repeated twice and the mean calculated concentration was taken for fitting. Specifically, HPV type 6 standard curve slope was 0.996, R 2 was 0.9950, HPV11 standard curve slope was 0.9448, R 2 was 0.9980, HPV16 standard curve slope was 0.9599, R 2 was 0.9985, HPV18 standard curve slope was 0.9885, R 2 was 0.9990, HPV31 standard curve slope was 0.9942, R 2 was 0.9984, HPV33 standard curve slope was 0.9935, R 2 was 0.9988, HPV45 standard curve slope was 0.9750, R 2 was 0.9984, HPV52 standard curve slope was 0.9649, R 2 was 0.9987, HPV58 standard curve slope was 1.008, R 2 was 0.9990 (FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3I). HPV type 6, HPV11, HPV16, HPV33 and HPV18, HPV31, HPV45 and HPV52 are in linear range of 5-5000ng/ml and HPV58 is in linear range of 5-10000ng/ml, which shows that the method has good detection performance.
Example 6 relative accuracy
The relative bias of each potency level relative potency measurement should be within + -12%, and the corresponding logarithm of potency measurement (ordinate) is linearly regressed with the logarithm of potency theory (abscissa), the slope of the regression equation should be withinWithin the range.
And (3) verifying the result, namely calculating the relative bias of the relative titer measured value of each titer level and the confidence interval thereof according to a calculation formula under the relative accuracy evaluation method item in the method of 9401 version 2020 of Chinese pharmacopoeia and the basic elements verified by the method, wherein the result is shown in tables 12-20. The relative bias is within + -10%. The logarithm of the theoretical value of the titer (abscissa) is used for carrying out linear regression on the logarithm of the corresponding measured value of the titer (ordinate).
And (3) carrying out quality control on the five concentrations, calculating relative other titer concentrations by taking the concentration near OD1.0 as 100%, wherein the average value of the relative bias in the nine types of reagents is within a range of +/-10%, and the average value of the relative bias meets the requirements. HPV type 6 standard curve slope was 0.9775, HPV11 standard curve slope was 0.9455, HPV16 standard curve slope was 0.9481, HPV16 standard curve slope was 0.9985, HPV18 standard curve slope was 0.9985, HPV31 standard curve slope was 0.9758, HPV33 standard curve slope was 0.9733, HPV45 standard curve slope was 0.9946, HPV52 standard curve slope was 1.028, HPV58 standard curve slope was 1.008.
TABLE 12 HPV6 type relative accuracy
TABLE 13 HPV11 relative accuracy
TABLE 14 HPV16 type relative accuracy
TABLE 15 relative accuracy of HPV18 type
TABLE 16 HPV31 relative accuracy
TABLE 17 HPV33 relative accuracy
TABLE 18 HPV45 relative accuracy
TABLE 19 relative accuracy of HPV52 type
TABLE 20 HPV58 relative accuracy
EXAMPLE 7 intermediate precision
Acceptable criteria the geometric coefficient of variation (GCV,%) of each potency level relative to the potency measurement should be no greater than 20%.
The verification result is that the in vitro potency part is calculated according to the four 9401 guidelines of pharmacopoeia 2020 edition, the precision of the relative potency determination method is generally expressed by Geometric Standard Deviation (GSD) or geometric variation coefficient (GCV,%) and is evaluated by adopting an analysis of variance (ANONA). The geometric standard deviation, geometric coefficient of variation, and upper confidence limit for each potency level versus potency measurement were compared and the results are shown in tables 21-29. The geometric coefficient of variation for each potency level relative to potency measurement is less than 20%.
Nine types of reagents all passed this standard.
TABLE 21 intermediate precision of HPV6
Table 22 HPV11 intermediate precision
TABLE 23 intermediate precision of HPV16
Table 24 intermediate precision of HPV18 type
Table 25 intermediate precision of HPV31 type
Table 26 HPV33 intermediate precision
Table 27 HPV45 intermediate precision
Table 28 intermediate precision of HPV52 type
Table 29 HPV58 intermediate precision
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