HPV-related protein monoclonal antibody and its application

Abstract

The present invention discloses a monoclonal antibody that specifically binds to HPV16 type L2 protein, the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, and the amino acid sequence of the HPV16 type L2 protein that the monoclonal antibody specifically binds to is GIDVDNTLYFSSNDNSINIA. The present invention also discloses a kit for detecting HPV16 type L2 protein antibodies, the kit comprising an effective amount of the monoclonal antibody and an effective amount of HPV16 type L2 protein; and a matching reagent for detecting HPV16 type L2 protein. The monoclonal antibody screened by the present invention has good sensitivity and specificity for HPV16 type L2 protein, and the HPV 16 type L2 protein antibody detection kit prepared on this basis also has good sensitivity, specificity and stability, and is suitable for large-scale detection of HPV16 type L2 protein antibodies.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody against HPV-related proteins and an application thereof. Background Art

[0002] Human papillomavirus (HPV) belongs to the Papillomaviridae family. It is a small molecular, non-enveloped circular double-stranded DNA virus with a genome length of approximately 8000bp (between 6800 and 8000bp). There are more than 200 subtypes, and about 40 types can cause female reproductive tract infections. Among them, HPV16 and 18 are the main types of cervical cancer infection worldwide, with infection rates of approximately 50% and 20%, respectively.

[0003] The HPV genome is divided into three functional regions, namely the early transcription region (E region), the late transcription region (L region) and the upstream regulatory region (URR region). The early region (E region) encodes genes necessary for the viral cycle and plays an important role in cell transformation (E1, E2, E4, E5, E6 and E7); the late region (L region): encodes the major coat protein L1 and the minor coat protein L2 of the virus particles; the upstream regulatory region (URR region), also known as the long control region (LCR), is a non-coding region containing replication origins and transcription factor binding sites, which regulates DNA replication by controlling viral gene transcription.

[0004] At present, the prevention and control of HPV is still mainly based on vaccine immunization. The HPV vaccines that have been launched include bivalent, quadrivalent, and nonavalent. All vaccines use VLPs assembled from HPV L1 protein and / or L2 protein as immunogens, where the "valence" corresponds to different types of HPV, such as bivalent for HPV16 and 18 vaccines.

[0005] There are many detection kits for HPVL1 protein on the market, but there are few detection kits for HPVL2, one of the immunogens. Therefore, it is urgent to develop a monoclonal antibody or detection kit that can detect HPVL2. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, the present invention aims to provide a monoclonal antibody that specifically binds to the HPVL2 protein and a detection kit for the HPVL2 protein antibody established on the basis of the monoclonal antibody.

[0007] Therefore, in one aspect, the present invention provides a monoclonal antibody that specifically binds to HPV type 16 L2 protein, and the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] Preferably, the amino acid sequence of the HPV type 16 L2 protein specifically bound by the monoclonal antibody of the present invention is GIDVDNTLYFSSNDNSINIA.

[0009] In another aspect, the present invention also provides a kit for detecting HPV 16 type L2 protein antibodies, the kit comprising an effective amount of the monoclonal antibody and an effective amount of HPV 16 type L2 protein; and supporting reagents for HPV 16 type L2 protein detection.

[0010] Preferably, the kit of the present invention is a blocking ELISA kit, which comprises an antigen-coated plate, a positive control, a negative control, a sample diluent, a 25× concentrated washing solution, an enzyme-labeled antibody, a substrate solution and a stop solution.

[0011] Preferably, the antigen-coated plate of the present invention is coated with HPV type 16 L2 protein at a coating concentration of 1 μg / ml.

[0012] Preferably, the positive control described in the present invention is the monoclonal antibody prepared in the present invention, and its use concentration is 1:5000 dilution.

[0013] Preferably, the enzyme-labeled antibody of the present invention is an HRP-labeled monoclonal antibody prepared by the present invention, and its use concentration is 1:20000 times diluted.

[0014] In another aspect, the present invention also provides a use of the monoclonal antibody in preparing a detection reagent for HPV type 16 L2 protein.

[0015] In another aspect, the present invention also provides a use of the kit in detecting HPV 16 type L2 protein antibodies.

[0016] The monoclonal antibodies screened by the present invention have good sensitivity and specificity for HPV type 16 L2 protein; and the HPV type 16 L2 protein antibody detection kit prepared on this basis also has good sensitivity, specificity and stability, and is suitable for large-scale detection of HPV type 16 L2 protein antibodies.

[0017] In addition, the monoclonal antibody against HPV 16 L2 protein screened by the present invention can not only be used to prepare an HPV 16 L2 protein antibody detection kit, but is also suitable for the detection of HPV 16 L2 protein (such as western blot detection, immunohistochemistry detection, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Comparison results of the amino acid sequences of L2 proteins of HPV types 11 / 16 / 18 / 6. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0020] Example 1: Preparation of HPV L2 protein

[0021] By comparing the amino acid sequences of L2 proteins of HPV 11 / 16 / 18 / 6, the results are as follows Figure 1 As shown in Table 1. The amino acid homology of the four common HPV L2 proteins is more than 60%, indicating a high degree of homology. In order to detect antibodies to different types of HPV, it is necessary to design highly specific monoclonal antibodies for the detection of different types of HPV. This study takes the most common HPV type 16 as the research object and designs monoclonal antibodies against the HPV type 16 L2 protein.

[0022] Table 1 Comparison of amino acid sequences of L2 proteins of HPV 11 / 16 / 18 / 6

[0023]

[0024] according to Figure 1 Based on the results, a highly specific antigen epitope peptide was designed for the amino acid sequence of HPV 16 L2 protein and commissioned for synthesis for subsequent experiments. The details are shown in Table 2.

[0025] Table 2 HPV 16 L2 protein antigen epitope peptides

[0026] Serial number Amino acid sequence ELISA titer 1 TRPPTADTLAPV <![CDATA[1:10 2 ]]> 2 PTPAETGGHFTLSSSTS <![CDATA[1:10 5 ]]> 3 GIDVDNTLYFSSNDNSINIA <![CDATA[1:10 6 ]]>

[0027] HPV 16 L2 protein was prepared using conventional protein preparation methods, which is an important raw material for the subsequent preparation of monoclonal antibodies and kits. For methods, please refer to the literature (e.g. Li Lingling. Expression and cross-reaction characteristics of HPV16 L2 full-length and its amino-terminal (1-200) prokaryotic fusion protein [D]. Wenzhou Medical College, 2010.).

[0028] Example 2: Preparation of monoclonal antibodies against HPV L2 protein antigen epitope polypeptides

[0029] Screening of antigen epitope polypeptides: The three antigen epitope polypeptides in Example 1 were injected intramuscularly into mice, 100 μg / 0.2 ml / mouse / time, and then boosted with the same dose 2 weeks later. Mouse serum was collected 2 weeks after the second immunization for ELISA antibody titer detection to screen the antigen epitope polypeptide with the highest antibody titer. The results showed (as shown in Table 2) that the titer of antigen epitope polypeptide 3 was the highest, and this antigen epitope polypeptide was selected as the preferred antigen for subsequent monoclonal antibody development.

[0030] The diluted antigen epitope polypeptide 3 and the adjuvant were mixed and emulsified in a ratio of 1:1 (wherein the concentration of antigen epitope polypeptide 3 in the vaccine was 100 μg / ml). Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent immunization.

[0031] During the first immunization, mice were inoculated intraperitoneally at 0.2 ml / mouse; 7 days later, mice were immunized for the second time via intraperitoneal inoculation at 0.2 ml / mouse; 7 days later, mice were directly immunized for the third time via intraperitoneal inoculation with antigen epitope peptide 3 at 100 μg / mouse; on the 3rd day after immunization, mouse spleen cells were taken and fused with mouse myeloma cells SP2 / 0 and cultured in HAT selective medium; 10 days later, cell supernatants were detected by indirect ELISA to screen positive hybridoma cells, from which a better monoclonal cell line was screened.

[0032] 8-10 week old Balb / c mice were intraperitoneally injected with 0.5 ml of pristane per mouse. 7-10 days later, 1-2×10 hybridoma cells were intraperitoneally injected into each mouse. 6 After 7 to 10 days, extract the mouse ascites, centrifuge at 12,000 r / min for 10 minutes at 2 to 8°C, and collect the supernatant. Purify the monoclonal antibody using a Protein G affinity chromatography column (or purify the monoclonal antibody using a conventional ammonium sulfate precipitation method) to obtain the monoclonal antibody, which is stored at -20°C for future use. Indirect ELISA detection, the titer of the monoclonal antibody is 1:10 6 This indicates that the prepared monoclonal antibody has good affinity to HPV type 16 L2 protein.

[0033] The heavy chain variable region and light chain variable region of the prepared monoclonal antibody were analyzed and determined. After detection and analysis, the heavy chain variable region and light chain variable region sequences of the monoclonal antibody were shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0034] Heavy chain variable region sequence (SEQ ID NO. 1): EVQVDKKLEP PELVKSTISM KISGSLGYTFTDYALLQKQSHGKNSVTVLVNPTWPSTIYN QSVAHPTLIV DCLVKTAYME FPESSEDSAVYPGCGDYGSSYGFAYWGQGT LVTVSAAKTT.

[0035] Light chain variable region sequence (SEQ ID NO. 2): MKLMTQTPLS LPVSLGDEQL TSCRSSNFYPSNGNTYKIDG SLQKPLNSWT LLIYKVSKDS TGVPERFSGS GSGTTLTLKI SRVKTSTGVYFCSFNRNTFGGGTKLEIKRADAAPTVSIFP.

[0036] Example 3: Specificity test of monoclonal antibodies

[0037] Different types of HPV L2 proteins were prepared according to the method of Example 1, and different types of HPV L2 proteins were used as coating antigens to perform indirect ELISA detection (see Example 4). The results showed that the ELISA antibody titers of the monoclonal antibodies of this study against different types of HPV L2 proteins (11, 18, 6) were all ≤1:10. This indicates that the monoclonal antibodies of this study have good specificity and can specifically recognize HPV 16 type L2 protein.

[0038] Example 4: Indirect ELISA steps

[0039] 1 Coating: Use HPV L2 protein (different types of HPV L2 proteins are prepared separately) to coat the ELISA plate. Use coating buffer (carbonate buffer) to dilute the HPV L2 protein to 1 μg / ml, mix well and add to the ELISA plate, 0.1 ml per well, and coat overnight at 2-8°C.

[0040] 2 Blocking: Wash three times with PBST buffer, and then block with PBST buffer containing 5% skim milk, 0.2 ml per well, incubate at 37°C for 2 h;

[0041] 3 Add samples: After washing three times with PBST buffer, add samples (cell culture supernatant is generally not diluted or diluted 10 times, and the purified monoclonal antibody is diluted 10 times in series, and the diluent is PBST buffer), 0.1 ml per well, 37 degrees for 1 hour;

[0042] 4 Add secondary antibody: After washing three times with PBST buffer, add HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) to each well; incubate at 37 degrees for 0.5h;

[0043] 5 Color development: After washing three times with PBST buffer, add TMB color development solution, 0.1 ml per well, at 37 degrees for 10 minutes;

[0044] 6 Stop: Add stop solution (2M sulfuric acid), 0.05 ml per well, and detect OD450nm value immediately after termination; when P / N value (sample OD450nm value / blank control OD value) > 2.1 and OD450nm value > 0.1, it is judged as positive, otherwise it is negative.

[0045] Example 5: Preparation of HPV 16 L2 Antibody Detection Kit (Blocking ELISA Kit)

[0046] 1. Preparation of antigen-coated plates:

[0047] Coating: Use HPV 16 type L2 protein to coat the ELISA plate. Use coating buffer (carbonate buffer) to dilute the HPV L2 protein to 1 μg / ml, mix well and add to the ELISA plate, 0.1 ml per well, and coat overnight at 2-8°C.

[0048] Blocking: Wash with PBST buffer for 3 times, and then block with PBST buffer containing 5% skim milk, 0.2 ml per well, incubate at 37°C for 2 hours. After drying, seal and store at 2-8°C for later use.

[0049] 2 Preparation of positive control: The monoclonal antibody prepared in Example 2 was diluted 1:5000 (the diluent was PBST buffer containing 2% BSA), and then quantitatively packaged as the positive control and stored at 2-8°C for later use.

[0050] 3. Preparation of negative control: quantitatively dispense PBST buffer containing 2% BSA, which is the negative control, and store it at 2-8°C for later use.

[0051] 4 Preparation of enzyme-labeled antibody: The monoclonal antibody prepared in Example 2 was labeled with HRP, and the specific labeling method was a modified sodium periodate method or a commercial kit. After labeling, it was diluted 1:20000 times with a diluent (PBST buffer containing 2% BSA), quantitatively divided, and stored at 2-8°C for later use.

[0052] 5 Kit Assembly

[0053] name quantity Antigen coated plates 2 plates (192 copies / box) Positive Control 1ml / tube Negative control 1ml / tube Sample diluent (PBST buffer containing 2% BSA) 12ml / bottle 25× concentrated washing buffer (25× PBST buffer) 30ml / bottle Enzyme-labeled antibody 25ml / bottle Substrate solution (TMB substrate solution) 25ml / bottle Stop solution (2M sulfuric acid) 12ml / bottle

[0054] 6 Test kit

[0055] (1) Sample addition: According to the number of samples to be tested, take the antigen-coated plate and add 50 μl of sample diluent to each well. Then add 50 μl of positive control, negative control and serum to be tested to the corresponding wells and vortex to mix.

[0056] (2) Incubation: Incubate at 37°C for 60 minutes.

[0057] (3) Washing: Shake off the liquid in the wells, add washing solution, 300 μl / well, and wash 3 to 5 times.

[0058] (4) Enzyme-labeled antibody incubation: Add enzyme-labeled antibody, 100 μl / well, and incubate at 37°C for 30 min.

[0059] (5) Washing: Same as (3).

[0060] (6) Color development: Add substrate solution, 100 μl / well, and incubate at 37°C in the dark for 10 min.

[0061] (7) Termination: Add stop solution, 50 μl / well, and shake to mix evenly.

[0062] (8) Reading: After adding the stop solution, immediately read the OD450nm value at a wavelength of 450nm.

[0063] (9) S / N value calculation: Calculate the S / N value according to the following calculation formula.

[0064]

[0065] (10)Judgment

[0066] Test establishment conditions: The OD450nm reading of each negative control well should be greater than 1.0 and the maximum difference between wells should be <0.3; the OD450nm reading of each positive control well should be <0.3.

[0067] Result determination: When the S / N value is greater than 0.6, it is judged as negative; when the S / N value is less than or equal to 0.6, it is judged as positive.

[0068] Example 6: Performance evaluation of HPV 16 type L2 antibody detection kit

[0069] 1. Sensitivity test

[0070] This kit was used to test 5 negative sera, and the results were all negative (S / N values ​​were 0.956, 0.964, 0.912, 0.925, and 0.935, respectively).

[0071] The kit was used to perform gradient dilution test on three positive sera, and the results showed (Table 3) that the dilution ratio of the three positive sera was 1:640, indicating that the kit had good sensitivity.

[0072] Table 3 Sensitivity test results of positive samples (S / N)

[0073]

[0074] 2 Specificity detection

[0075] The other types of HPV L2 proteins in Example 3 were used to prepare vaccines, and mice were immunized to prepare mouse serum (mouse titer was detected using the method of Example 4). The mouse serum was tested using this kit, and the results showed that the test results of this kit were all negative (as shown in Table 4), indicating that this kit has good specificity.

[0076] Table 4 Specificity test results (S / N value)

[0077]

[0078] 3. Repeatability test

[0079] The kit was used to test three different serum samples. The results showed (Table 5) that the intra-batch CV values ​​of the kit were all less than 5%, indicating that the kit had good intra-batch repeatability.

[0080] The same method was used to prepare three batches of the kit, and three different serum samples were tested to determine the batch reproducibility of the kit. The results showed (Table 6) that the batch reproducibility of the kit was also good (CV values ​​were all less than 10%).

[0081] Table 5 Intra-batch repeatability test results

[0082]

[0083] Table 6 Results of inter-batch repeatability test

[0084]

[0085] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A monoclonal antibody that specifically binds to HPV type 16 L2 protein, characterized in that: The sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the HPV 16 type L2 protein specifically bound by the monoclonal antibody is GIDVDNTLYFSSNDNSINIA.

3. A kit for detecting HPV type 16 L2 protein antibodies, characterized in that: The kit comprises an effective amount of the monoclonal antibody according to claim 1 and an effective amount of HPV 16 type L2 protein; and a supporting reagent for detecting HPV 16 type L2 protein.

4. The kit according to claim 3, characterized in that The kit is a blocking ELISA kit, which comprises an antigen-coated plate, a positive control, a negative control, a sample diluent, a 25× concentrated washing solution, an enzyme-labeled antibody, a substrate solution and a stop solution.

5. The kit according to claim 4, characterized in that The antigen coating plate is coated with HPV 16 type L2 protein, and the coating concentration is 1 μg / ml.

6. The kit according to claim 4, characterized in that The positive control is the monoclonal antibody of claim 1, and its use concentration is 1:5000 dilution.

7. The kit according to claim 4, characterized in that The enzyme-labeled antibody is the monoclonal antibody of claim 1 labeled with HRP, and its use concentration is 1:20000 times diluted.

8. Use of the monoclonal antibody according to claim 1 in preparing a detection reagent for HPV type 16 L2 protein.

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