Anti-rHPV68 L1 protein CD68 monoclonal antibody, detection kit for rHPV68 type protein antigen

Abstract

This application relates to the technical field of biological detection, and specifically discloses an anti-rHPV68 L1 protein CD68 monoclonal antibody and a detection kit for rHPV68 type protein antigen. This application discloses an anti-rHPV68 L1 protein CD68 monoclonal antibody, and the DNA sequence of the anti-rHPV68 L1 protein CD68 monoclonal antibody is shown as SEQ ID NO.1. Using the above anti-rHPV68 L1 protein CD68 monoclonal antibody, this application provides a TRFIA detection kit for the content of rHPV68 type protein antigen. When using this TRFIA detection kit to detect the content of rHPV68 type protein antigen, it has strong specificity, and the sensitivity, accuracy and precision of this detection method are relatively high.

Description

Technical Field

[0001] The present application relates to the technical field of biological detection, and particularly relates to a CD68 monoclonal antibody against rHPV68 L1 protein and a detection kit for rHPV68 type protein antigen. Background Art

[0002] Human papillomavirus (HPV) can cause squamous epithelial proliferation of human skin and mucous membranes, and can lead to a variety of benign and malignant lesions. Low-risk HPV infection may cause condyloma acuminata, and high-risk HPV infection may cause cervical lesions or even cervical cancer. HPV is a small spherical circular double-stranded DNA virus, and is classified according to the homology of DNA. There are more than 200 subtypes of HPV viruses, among which there are about 19 high-risk viruses such as HPV16, HPV18, HPV31, and HPV68. HPV68 belongs to Group 2A in the IARC carcinogen classification.

[0003] In the research and production of HPV vaccines, it is necessary to establish detection methods for identifying antigen content, in vitro relative potency, etc. Since monoclonal antibodies have high specificity and neutralizing activity, currently these detection methods generally use enzyme-linked immunosorbent assay (ELISA), and ELISA using monoclonal antibodies as detection antibodies is more common. However, the process of ELISA is relatively complex, requires higher quality of operators, and is greatly interfered by external conditions, resulting in lower specificity, sensitivity, and accuracy of the detection method.

[0004] Based on the above, there is an urgent need to develop a detection method with high specificity, high sensitivity, and high accuracy for determining HPV type protein antigen. Summary of the Invention

[0005] In order to improve the specificity, sensitivity, and accuracy of the detection method for rHPV68 type protein antigen content, the present application provides a CD68 monoclonal antibody against rHPV68 L1 protein and a detection kit for rHPV68 type protein antigen.

[0006] In the first aspect, the present application provides a CD68 monoclonal antibody against rHPV68 L1 protein, and the DNA sequence of the CD68 monoclonal antibody against rHPV68 L1 protein is shown as SEQ ID NO.1.

[0007] The function of an antibody depends on its surface sites that bind to an antigen, and antibodies exert specific functions due to different binding surface sites. Therefore, screening antibodies at specific sites can specifically recognize and detect specific subtypes of HPV protein antigens. The CD68 monoclonal antibody against rHPV68 L1 protein shown in the present application has high specificity for detecting rHPV68 type protein antigen.

[0008] In a second aspect, the present application provides the use of the above-mentioned anti-rHPV68 L1 protein CD68 monoclonal antibody in the field of detecting rHPV68 type protein.

[0009] In a third aspect, a TRFIA detection kit for the antigen content of rHPV68 type protein, the detection kit includes: the above-mentioned anti-rHPV68 L1 protein CD68 monoclonal antibody as the primary antibody; the anti-rHPV68 L1 protein CD68 monoclonal antibody labeled with lanthanide element ions as the secondary antibody; a standard sample of rHPV68 type protein antigen.

[0010] The time-resolved immunofluorescence (TRFIA) used in the present application is a non-isotope immunoassay technique. It labels antibodies with lanthanide elements, measures fluorescence using time-resolved techniques according to the luminescence characteristics of lanthanide element chelates, and resolves signals by detecting two parameters of wavelength and time simultaneously, which can effectively exclude the interference of non-specific fluorescence and greatly improve the sensitivity of analysis; and the TRFIA detection method has a low background and a high signal-to-noise ratio, which is beneficial to improving the sensitivity and accuracy of the detection method.

[0011] Through experimental analysis, it can be known that when the ELISA detection method is selected to detect the antigen content of rHPV68 type protein, the recovery rate of the detection result is quite different from 100%, and the coefficient of variation is relatively high; while when the TRFIA detection method used in the present application is selected to detect the antigen content of rHPV68 type protein, the recovery rate of the detection result is close to 100%, and the coefficient of variation is relatively low, indicating that the TRFIA detection method provided by the present application can effectively improve the accuracy and precision of the detection result. Therefore, the present application selects the TRFIA detection method to detect the antigen content of rHPV68 type protein.

[0012] Preferably, the detection kit further includes a primary antibody diluent and a secondary antibody diluent.

[0013] Further, the primary antibody diluent is a Tris-HCl buffer solution with a pH of 7.0 - 7.5 and a concentration of 40 - 60 mmol / L.

[0014] Further, the secondary antibody diluent is a Tris-HCl buffer solution containing 0.01 - 0.05% BSA, 0.01 - 0.1% Tween-20, 0.01 - 0.1% NaN3, with a pH of 7.5 - 8.0 and a concentration of 40 - 60 mmol / L.

[0015] Preferably, the concentration of the primary antibody is 2 - 4 μg / mL.

[0016] In a specific embodiment, the concentration of the primary antibody can be: 2 μg / mL, 3 μg / mL, 4 μg / mL.

[0017] In some specific embodiments, the concentration of the primary antibody can also be: 2-3 μg / mL, 3-4 μg / mL.

[0018] Through experimental analysis, it can be known that when the concentration of the primary antibody is controlled within the above range, the accuracy and precision of the detection results can be further improved. Therefore, the concentration of the primary antibody in this application is controlled within the above range.

[0019] Preferably, the concentration of the secondary antibody is 180-220 μg / mL.

[0020] In a specific embodiment, the concentration of the secondary antibody can be: 180 μg / mL, 200 μg / mL, 220 μg / mL.

[0021] In some specific embodiments, the concentration of the secondary antibody can also be: 180-200 μg / mL, 200-220 μg / mL.

[0022] Through experimental analysis, it can be known that when the concentration of the secondary antibody is controlled within the above range, the accuracy and precision of the detection results can be further improved. Therefore, the concentration of the secondary antibody in this application is controlled within the above range.

[0023] Preferably, the lanthanide ion is Eu 3+ , Dy 3+ , Tb 3+ , Sm 3+ or one or more of them.

[0024] Further, the lanthanide ion is Eu 3+ .

[0025] Preferably, the weight ratio of the lanthanide ion to the anti-rHPV68 L1 protein CD68 monoclonal antibody in the secondary antibody is (0.1-0.3):1.

[0026] In a specific embodiment, the weight ratio of the lanthanide ion to the anti-rHPV68 L1 protein CD68 monoclonal antibody can be 0.1:1, 0.2:1, 0.3:1.

[0027] In some specific embodiments, the weight ratio of the lanthanide ion to the anti-rHPV68 L1 protein CD68 monoclonal antibody can also be (0.1-0.2):1, (0.2-0.3):1.

[0028] Through experimental analysis, it can be known that when the weight ratio of the lanthanide element ion to the anti-rHPV68 L1 protein CD68 monoclonal antibody is within the above range, the accuracy and precision of the detection result can be further improved. Therefore, the present application controls the weight ratio of the lanthanide element ion to the anti-rHPV68 L1 protein CD68 monoclonal antibody within the above range.

[0029] Preferably, the detection kit further comprises a blocking solution and an enhancement solution.

[0030] Further, the blocking solution is selected from one or more of a BSA solution, a skim milk powder solution, and a horse serum albumin solution.

[0031] Through experimental analysis, it can be known that compared with the selection of using a skim milk powder solution or a horse serum albumin solution, the present application selects to use a BSA solution as the blocking solution, which can further improve the accuracy and precision of the detection result. Therefore, the present application selects to use a BSA solution as the blocking solution.

[0032] Further, the concentration of the BSA solution is 1-1.5 wt%.

[0033] In a specific embodiment, the concentration of the BSA solution can be: 1 wt%, 1.2 wt%, 1.5 wt%.

[0034] In some specific embodiments, the concentration of the BSA solution can also be: 1-1.2 wt%, 1.2-1.5 wt%.

[0035] Through experimental analysis, it can be known that when the concentration of the BSA solution is controlled within the above range, the accuracy and precision of the detection result can be further improved. Therefore, the present application controls the concentration of the BSA solution within the above range.

[0036] Preferably, the enhancement solution is an acetic acid-phthalic acid buffer solution containing 40-60 μmol / L TOPO, 10-20 μmol / L β-NAT, 0.1% Triton X-100, with a pH of 7.5-8.0 and a concentration of 80-100 mmol / L. Further, the detection kit further comprises a washing solution and a protein sample diluent.

[0037] Further, the washing solution is a Tris-HCl buffer solution containing 0.5-1% NaCl, 0.01-0.05% NaN3, 0.05-0.1% Tween 20, with a pH of 7.5-8.0 and a concentration of 50 mmol / L.

[0038] Further, the protein sample diluent is a Tris-HCl buffer solution containing 0.01%-0.1% NaN3, 0.1%-0.3% bovine serum albumin, with a pH of 7.5-8.0 and a concentration of 40-60 mmol / L.

[0039] Fourthly, the present application provides a TRFIA detection method for the content of rHPV68 protein antigen. The TRFIA detection method uses the above detection kit to detect the content of rHPV68 protein antigen, and specifically includes the following steps:

[0040] S1: Coat the enzyme-linked immunosorbent assay (ELISA) plate with the primary antibody and add a blocking solution for blocking to obtain a solid-phase monoclonal antibody material.

[0041] S2: Label the anti-rHPV68 L1 protein CD68 monoclonal antibody with lanthanide ions to prepare a secondary antibody.

[0042] S3: Add a standard sample or a sample to be tested and the secondary antibody to the solid-phase monoclonal antibody material in S1, and then add an enhancement solution for incubation to obtain an ELISA plate to be tested.

[0043] S4: Perform fluorescence measurement on the ELISA plate to be tested to obtain the content of rHPV68 protein antigen in the sample to be tested.

[0044] In summary, the technical solution of the present application has the following effects:

[0045] The present application uses the anti-rHPV68 L1 protein CD68 monoclonal antibody and the TRFIA detection method to specifically detect the rHPV68 protein antigen, and can obtain a standard curve with strong correlation, which is beneficial to quickly obtain the content of the rHPV68 protein antigen.

[0046] When using the detection method provided by the present application to detect the content of rHPV68 protein antigen, it has strong specificity, and high sensitivity, accuracy and precision. Description of the Drawings

[0047] Figure 1 It is a schematic diagram of the principle of the TRFIA detection method for the content of rHPV68 protein antigen in the present application.

[0048] Figure 2 It is the "concentration-fluorescence value" standard curve provided in Example 3 of the present application. Detailed Embodiments

[0049] In a first aspect, the present application provides an anti-rHPV68 L1 protein CD68 monoclonal antibody, and the DNA sequence of the anti-rHPV68 L1 protein CD68 monoclonal antibody is as shown in SEQ ID NO.1.

[0050] In a second aspect, the present application provides the use of the above anti-rHPV68 L1 protein CD68 monoclonal antibody in the field of detecting rHPV68 type protein.

[0051] In a third aspect, the present application provides a TRFIA detection kit for the antigen content of rHPV68 type protein, including: the above anti-rHPV68 L1 protein CD68 monoclonal antibody as the primary antibody, the anti-rHPV68 L1 protein CD68 monoclonal antibody labeled with lanthanide element ions as the secondary antibody, the rHPV68 type protein antigen standard sample, the blocking solution, the washing solution, and the enhancement solution.

[0052] Among them, the concentration of the primary antibody is 2 - 4 μg / mL, and the concentration of the secondary antibody is 180 - 220 μg / mL.

[0053] Specifically, the lanthanide element ion is Eu 3+ , Dy 3+ , Tb 3+ , Sm 3+ or one or more of them; further, the lanthanide element ion is Eu 3+ .

[0054] At the same time, the weight ratio of the lanthanide element ion to the anti-rHPV68 L1 protein CD68 monoclonal antibody in the secondary antibody is (0.1 - 0.3):1.

[0055] In addition, the blocking solution is selected from one or more of BSA solution, skim milk powder solution, and horse serum albumin solution.

[0056] Further, the concentration of the BSA solution is 1 - 1.5 wt%.

[0057] Further, the enhancement solution is an acetic acid - phthalic acid buffer solution containing 40 - 60 μmol / L TOPO, 10 - 20 μmol / L β-NAT, 0.1% Triton X-100, with a pH of 7.5 - 8.0 and a concentration of 80 - 100 mmol / L.

[0058] In a fourth aspect, the present application provides a method for detecting the antigen content of rHPV68 type protein using the above detection kit, specifically including the following steps:

[0059] S1: Preparation of the solid-phase monoclonal antibody material

[0060] Using a Tris-HCl buffer solution with a pH of 7.0 - 7.5 and a concentration of 40 - 60 mmol / L as the primary antibody diluent, dilute the concentration of the primary antibody to 2 - 4 μg / mL, and add 100 μL per well to a 96-well enzyme-linked immunosorbent assay (ELISA) plate. After sealing the plate with a sealing film, incubate overnight at 4°C; then use a Tris-HCl buffer solution containing 0.5 - 1% NaCl, 0.01 - 0.05% NaN₃, 0.05 - 0.1% Tween 20, with a pH of 7.5 - 8.0 and a concentration of 50 mmol / L as the washing solution. Wash the ELISA plate 5 times with 300 μL per well of the washing solution, and finally pat dry the detection plate to obtain the coated ELISA plate.

[0061] Add 300 μL per well of the blocking solution to the coated ELISA plate and incubate at 37°C for 2 h; then wash the plate 5 times with 300 μL per well of the washing solution, and finally pat dry the detection plate. Store it at a temperature below -20°C to obtain the blocked ELISA plate, which is the solid-phase monoclonal antibody material. Among them, the blocking solution is one or more of a BSA solution, a skim milk powder solution, and a horse serum albumin solution.

[0062] S2: Eu 3+ Preparation of Eu-labeled anti-rHPV68 L1 protein CD68 monoclonal antibody

[0063] Use a Na₂CO₃–NaHCO₃ buffer solution containing 150 - 160 mmol / L NaCl, with a pH of 8.2 - 8.8 and a concentration of 40 - 60 mmol / L as the labeling solution; add the labeling solution to the anti-rHPV68 L1 protein CD68 monoclonal antibody, and then mix it with Eu 3+ -chelate; gently shake the mixture at room temperature and incubate for 18 h; then purify the mixture with a Sephadex G50 column to obtain Eu 3+ Labeled detection antibody, which is the secondary antibody, and store it at a temperature below -20°C.

[0064] S3: Preparation of the enzyme-linked immunosorbent assay (ELISA) plate to be tested

[0065] Use a Tris-HCl buffer solution containing 0.01% - 0.1% NaN₃, 0.1% - 0.3% bovine serum albumin, with a pH of 7.5 - 8.0 and a concentration of 40 - 60 mmol / L as the protein sample diluent. Dilute the reference sample to be tested to obtain standard solutions with different concentrations, and dilute the sample to be tested. Add the diluted standard solutions and sample solutions to the blocked ELISA plate at 100 μL per well. After sealing the plate with a sealing film, incubate with shaking at room temperature for 1 h; wash the ELISA plate 4 times with 300 μL per well of the washing solution, and finally pat dry the detection plate to obtain the ELISA plate containing the sample to be tested.

[0066] Using a Tris-HCl buffer solution containing 0.01 - 0.05% BSA, 0.01 - 0.1% Tween-20, 0.01 - 0.1% NaN3, with a pH of 7.5 - 8.0 and a concentration of 40 - 60 mmol / L as the secondary antibody diluent, dilute the concentration of the secondary antibody to a concentration of 100 - 300 μg / mL, and add the diluted secondary antibody to the enzyme-linked immunosorbent assay (ELISA) plate containing the sample to be tested at 100 μL / well. After sealing the plate with a sealing film, incubate it with shaking at room temperature for 1 h; wash the ELISA plate 6 times with 300 μL / well of washing solution, and finally pat dry the detection plate to obtain an ELISA plate containing the secondary antibody.

[0067] Using an acetic acid - phthalic acid buffer solution containing 40 - 60 μmol / L TOPO, 10 - 20 μmol / L β-NAT, 0.1% Triton X-100, with a pH of 7.5 - 8.0 and a concentration of 80 - 100 mmol / L as the enhancer, add it to the ELISA plate containing the secondary antibody at 100 μL / well, and incubate it with shaking at room temperature for 5 min to obtain the ELISA plate to be tested.

[0068] S4: Use a semi-automatic Victor3TM 1420 multi-label detector to measure, draw a standard curve using the detection results of standard solutions with different concentrations, and then use this standard curve to calculate the content of the antigen in the sample to be tested.

[0069] The following further describes the present application in detail in combination with Preparation Examples 1 - 8, Examples 1 - 19, Comparative Examples, and performance detection tests. These examples should not be construed as limiting the scope claimed in the present application.

[0070] Preparation Example

[0071] Preparation Example 1

[0072] This preparation example provides a CD68 monoclonal antibody against the rHPV68 L1 protein.

[0073] The preparation method of the CD68 monoclonal antibody against the rHPV68 L1 protein in this preparation example is as follows:

[0074] S1: Thoroughly mix the rHPV68 protein antigen solution and Freund's complete adjuvant to serve as the primary immunizing agent; thoroughly mix the rHPV68 protein antigen solution and Freund's incomplete adjuvant to serve as the booster immunizing agent; among them, Freund's complete adjuvant and incomplete adjuvant are purchased from Sigma Corporation.

[0075] S2: Immunize animals: Select 6 female BLAB / c mice at 7 weeks old and immunize them. First immunization: Inject the basic immunizing agent subcutaneously into the back of the mice. Second immunization: After 14 days, inject the booster immunizing agent subcutaneously into the bilateral inguinal regions of the mice. Third immunization: After 14 days, inject the booster immunizing agent subcutaneously into the posterior parts of the bilateral shoulders and the abdomen of the mice. Final immunization: After 7 days, collect blood from the tail vein of the mice, detect the antibody titer in the serum by ELISA. When the antibody titer reaches 1:10000, inject the booster immunizing agent into the abdominal cavity of the mice.

[0076] S3: Prepare hybridoma cells: Collect blood 3 days after the final immunization, aseptically take the spleens of the mice and prepare spleen cell suspensions; fuse the mouse spleen cell suspensions with mouse myeloma SP2 / 0 cells at a ratio of 1:1 to form hybridoma cells. Gently suspend the cells with HAT culture medium (purchased from Sigma Company) and divide the cells into 96-well plates at 200 μL per well; observe the cell fusion situation after culturing for three days, change the culture medium, and continuously culture until clones are formed. Change the culture medium 7 days after fusion. Use the indirect ELISA method to screen the cells that produce anti-rHPV68 L1 protein CD68 monoclonal antibodies, and then perform subcloning on the positive wells with high titers by the limiting dilution method until the cell positive rate reaches 100%. Finally, obtain the cells that stably secrete anti-rHPV68 L1 protein CD68 monoclonal antibodies, and entrust GenScript Biotech Corporation to perform DNA sequence determination on the obtained anti-rHPV68 L1 protein CD68 monoclonal antibodies, as shown in SEQ ID NO.1.

[0077] S4: Plasmid extraction: According to the DNA sequence shown in SEQ ID NO.1, entrust GenScript Biotech Corporation to synthesize the HPV68 L1 gene cloning and expression plasmid, and use a plasmid extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to extract the plasmid to obtain the heavy chain plasmid and the light chain plasmid. Take 100 mg of the extracted plasmid, add 120 mg of the endotoxin removal solution, and incubate on ice for 45 min; and use an endotoxin detection kit to detect and confirm that the endotoxin in the plasmid is less than 0.15 EU, that is, the transfection-grade plasmid is obtained. Add the above-obtained transfection-grade plasmid to 100 mL of TE buffer.

[0078] S5: Cell transfection: Prepare 293FT cells and control the cell confluence at 60%. Mix the plasmid and lipo2000 in proportion and let it stand for 20 min. Then slowly add the mixture of the plasmid and lipo2000 to the cell culture flask, mix well, and place it in a constant temperature cell incubator at 37 °C and 5% CO2 for cell transfection.

[0079] S6: Purify the antibody protein: Centrifuge the culture medium of the transfected 293FT cells, filter the cell supernatant with a 0.22-μm filter membrane, dialyze overnight at 4°C in a carbonate buffer with a pH of 9.6, and then purify it with a Protein G column to obtain the anti-rHPV68 L1 protein CD68 monoclonal antibody.

[0080] The DNA sequence of the anti-rHPV68 L1 protein CD68 monoclonal antibody prepared in this Preparation Example is as shown in SEQ ID NO.1.

[0081] Preparation Examples 2-6

[0082] Preparation Examples 2-6 respectively provide a blocking solution.

[0083] The differences among the above Preparation Examples lie in: the concentration of BSA in the blocking solution. Specifically as shown in Table 1.

[0084] The preparation method of the above Preparation Examples is: According to Table 1, weigh BSA and dissolve it fully in 95 g of a Na2CO3–NaHCO3 buffer with a pH of 9.6 and a concentration of 50 mmol / L, and make up the weight to 100 g with deionized water to prepare the blocking solution.

[0085] Table 1 The addition amount of BSA in the blocking solution in Preparation Examples 1-5

[0086] Preparation Example Amount of BSA added (g) Concentration of BSA (wt%) 2 0.8 0.8 3 1 1 4 1.2 1.2 5 1.5 1.5 6 1.7 1.7

[0087] Preparation Example 7

[0088] This Preparation Example provides a blocking solution.

[0089] The preparation method of the blocking solution in this Preparation Example is: Weigh 1.2 g of skim milk powder and dissolve it fully in 95 g of a Na2CO3–NaHCO3 buffer with a pH of 9.6 and a concentration of 50 mmol / L, and make up the weight to 100 g with deionized water to prepare the blocking solution.

[0090] Preparation Example 8

[0091] This Preparation Example provides a blocking solution.

[0092] The preparation method of the blocking solution in this Preparation Example is: Weigh 1.2 g of horse serum albumin and dissolve it fully in 95 g of a Na2CO3–NaHCO3 buffer with a pH of 9.6 and a concentration of 50 mmol / L, and make up the weight to 100 g with deionized water to prepare the blocking solution.

[0093] Examples

[0094] Examples 1-7

[0095] Examples 1-7 respectively provide a method for detecting the content of rHPV68 protein antigen.

[0096] The differences among the above examples are as follows: the types of blocking solutions in step S2 are different. Specifically, as shown in Table 2.

[0097] The schematic diagram of the principle of the detection method of the above examples is as Figure 1 shown, and specifically includes the following steps:

[0098] S1: Preparation of solid-phase monoclonal antibody material

[0099] Use the anti-rHPV68 L1 protein CD68 monoclonal antibody as the primary antibody, use the Tris-HCl buffer solution with pH = 7.2 and a concentration of 50 mmol / L as the primary antibody diluent, dilute the concentration of the primary antibody to 3 μg / mL, and add 100 μL / well to a 96-well enzyme-linked immunosorbent assay (ELISA) plate. After sealing the plate with a sealing film, incubate overnight at 4 °C; then use the Tris-HCl buffer solution containing 0.84% NaCl, 0.01% NaN3, 0.06% Tween 20, with pH = 7.8 and a concentration of 50 mmol / L as the washing solution. Wash the ELISA plate 5 times with 300 μL / well of the washing solution, and finally pat dry the detection plate to obtain the coated ELISA plate.

[0100] Add the blocking solution to the coated ELISA plate at 300 μL / well and incubate at 37 °C for 2 h; then wash the plate 5 times with 300 μL / well of the washing solution, and finally pat dry the detection plate. Store it at a temperature below -20 °C to obtain the blocked ELISA plate, which is the solid-phase monoclonal antibody material.

[0101] S2: Preparation of Eu 3+ -labeled anti-rHPV68 L1 protein CD68 monoclonal antibody

[0102] Use the Na2CO3–NaHCO3 buffer solution containing 155 mmol / L NaCl, with pH = 8.5 and a concentration of 50 mmol / L as the labeling solution; add 1 mg of anti-rHPV68 L1 protein CD68 monoclonal antibody to 1 mL of the labeling solution, and then mix it with 0.2 mg of Eu 3+ -chelate; gently shake the mixture at room temperature and incubate for 18 h; then purify the mixture with a Sephadex G50 column to obtain Eu 3+ -labeled anti-rHPV68 L1 protein CD68 monoclonal antibody, which is the secondary antibody, and store it at a temperature below -20 °C.

[0103] S3: Preparation of the enzyme-linked immunosorbent assay (ELISA) plate to be tested

[0104] Add the sample to be tested: Use a Tris-HCl buffer solution containing 0.05% NaN3, 0.2% bovine serum albumin, with a pH of 7.8 and a concentration of 50 mmol / L as the protein sample diluent. Add the diluted standard solution and the sample solution to be tested into the sealed enzyme-linked immunosorbent assay (ELISA) plate at 100 μL / well. After sealing the plate with a sealing film, incubate it with shaking at room temperature for 1 h. Wash the ELISA plate 4 times with 300 μL / well of washing solution, and finally pat dry the detection plate to obtain an ELISA plate containing the sample to be tested.

[0105] Add the secondary antibody: Use a Tris-HCl buffer solution containing 0.03% BSA, 0.05% Tween-20, 0.05% NaN3, with a pH of 7.8 and a concentration of 50 mmol / L as the secondary antibody diluent. Dilute the secondary antibody to a concentration of 200 μg / mL. Add the diluted secondary antibody to the ELISA plate containing the sample to be tested at 100 μL / well. After sealing the plate with a sealing film, incubate it with shaking at room temperature for 1 h. Wash the ELISA plate 6 times with 300 μL / well of washing solution, and finally pat dry the detection plate to obtain an ELISA plate containing the secondary antibody.

[0106] Add the enhancer: Use an acetic acid-phthalic acid buffer solution containing 50 μmol / L TOPO, 15 μmol / L β-NAT, 0.1% Triton X-100, with a pH of 7.8 and a concentration of 100 mmol / L as the enhancer. Add it to the ELISA plate containing the secondary antibody at 100 μL / well, and incubate it with shaking at room temperature for 5 min to obtain the ELISA plate to be tested.

[0107] S4: Determination of the content of rHPV68 protein antigen in the sample to be tested

[0108] Use a semi-automatic Victor3 TM 1420 multi-label detector to detect the ELISA plate to be tested, measure the fluorescence signal values of standard solutions with different concentrations, and make a standard curve with the double logarithm of concentration and fluorescence signal value.

[0109] According to the standard curve, calculate the content of rHPV68 protein antigen in the sample to be tested.

[0110] Table 2 Types of blocking solutions in Examples 1-7

[0111]

[0112] Examples 8-11

[0113] Examples 8-11 respectively provide a method for detecting the content of rHPV68 protein antigen.

[0114] The differences between the above embodiments and Embodiment 3 are as follows: the weight ratio of the lanthanide ion to the anti-rHPV68 L1 protein CD68 monoclonal antibody. Specifically, as shown in Table 3.

[0115] Table 3 Weight ratios of lanthanide ions to anti-rHPV68 L1 protein CD68 monoclonal antibodies in Embodiments 3, 8 - 12

[0116]

[0117] Embodiments 12 - 19

[0118] Embodiments 12 - 19 respectively provide a method for detecting the content of rHPV68 type protein antigen.

[0119] The differences between the above embodiments and Embodiment 3 are as follows: the concentration of the primary antibody and the concentration of the secondary antibody. Specifically, as shown in Table 4.

[0120] Table 4 Concentrations of the primary antibody and the secondary antibody in Embodiments 3, 12 - 19

[0121]

[0122] Comparative Example

[0123] The comparative example provides an ELISA detection method for the content of rHPV68 type protein antigen.

[0124] The specific steps of the detection method in this comparative example are as follows:

[0125] S1: Preparation of the blocked enzyme-linked immunosorbent assay (ELISA) plate

[0126] Using the anti-rHPV68 L1 protein CD68 monoclonal antibody as the primary antibody, dilute the primary antibody to 2 μg / mL with the coating solution and add 100 μL / well to a 96-well ELISA plate. After sealing the plate with a sealing film, incubate overnight at 4°C; the preparation method of the coating solution is: weigh 6.4 g of sodium carbonate (Na2CO3) and 11.7 g of sodium bicarbonate (NaHCO3), dissolve and dilute to 1000 mL with purified water. Dilute 4-fold with purified water before use.

[0127] Wash the plate 2 times with 300 μL / well of the washing solution, pat dry the detection plate for the last time, and then add the blocking solution provided in Preparation Example 4 to the coated ELISA plate at 300 μL / well and incubate at 37°C for 1.5 h.

[0128] S2: Preparation of the enzyme-linked immunosorbent assay (ELISA) plate to be tested

[0129] Add the sample to be tested: After washing the enzyme-linked immunosorbent assay (ELISA) plate that has completed the blocking step once with 300 μL / well of washing solution and patting it dry, use PBS solution containing 1% BSA and 0.05% Tween-20 as the sample diluent. Add the diluted standard solution and the sample solution to be tested to the ELISA plate at 100 μL / well. Seal the plate with a sealing film and incubate it with shaking at room temperature for 1 h.

[0130] Add the secondary antibody: Wash the ELISA plate three times with 300 μL / well of washing solution, and finally pat the detection plate dry. Use PBS solution containing 3% BSA and 0.05% Tween-20 as the diluent. Dilute the anti-rHPV68 L1 protein CD68 monoclonal antibody to a concentration of 0.05 μg / mL as the secondary antibody, and add it to the ELISA plate containing the sample to be tested at 100 μL / well. Seal the plate with a sealing film and incubate it with shaking at room temperature for 1 h.

[0131] Add the chromogenic solution: Wash the ELISA plate five times with 300 μL / well of washing solution, and finally pat the detection plate dry. Add TMB chromogenic solution to the ELISA plate at 100 μL / well. After incubating it in the dark at room temperature for 15 min, add 50 μL / well of 1 mol / L sulfuric acid to terminate the reaction, and obtain the ELISA plate to be tested.

[0132] S3: Determination of the content of rHPV68 protein antigen in the sample to be tested

[0133] Put the ELISA plate to be tested into an ELISA reader, and read the OD450 value and perform data analysis.

[0134] Performance detection test

[0135] (1) Accuracy and precision of the detection method when detecting the sample to be tested with the same concentration

[0136] Detection method: Use the standard solution with a theoretical concentration of 35.92 ng / mL as the sample to be tested. Use the detection methods provided in Examples 1-19 and the comparative example to repeatedly measure the content of rHPV68 protein antigen in the above sample to be tested 6 times, perform statistical processing, and calculate the recovery rate and coefficient of variation of the detection method. Use the above method to investigate the accuracy and precision of the detection methods provided in Examples 1-19 and the comparative example when detecting the sample to be tested with the same concentration.

[0137] Among them, the recovery rate = (average measured concentration M / theoretical concentration) × 100%; the coefficient of variation = (standard deviation SD / average measured concentration M) × 100%.

[0138] Detection results: As shown in Table 5.

[0139] Table 5 Accuracy and precision of the detection method when detecting the sample to be tested with the same concentration

[0140]

[0141]

[0142] Combined with Table 5, by comparing the detection results of Examples 1-19 with those of the comparative examples, the present application uses the provided anti-rHPV68L1 protein CD68 monoclonal antibody and the TRFIA detection method to detect the content of rHPV68 protein antigen. When the concentration of the sample to be tested is controlled to be constant, the recovery rate of the detection results of the present application can be controlled within the range of 93.76-102.28%, and the coefficient of variation is less than 3.86%. The above detection results indicate that the TRFIA detection method for the content of rHPV68 protein antigen provided by the present application can obtain high accuracy and precision.

[0143] By comparing the detection results of Example 3 with those of the comparative example, it can be seen that when the ELISA detection method is selected to detect the content of rHPV68 protein antigen, the recovery rate of the detection results is 87.81%, and the coefficient of variation is 18.64%; while when the TRFIA detection method provided by the present application is selected to detect the content of rHPV68 protein antigen, the recovery rate of the detection results is 100.03%, and the coefficient of variation is 0.95%; the above detection results indicate that the TRFIA detection method provided by the present application can effectively improve the accuracy and precision of the detection results. Therefore, the present application selects the TRFIA detection method to detect the content of rHPV68 protein antigen.

[0144] By comparing the detection results of Example 3 with those of Examples 6-7, compared with the selection of using skim milk powder solution or horse serum albumin solution, the present application selects to use BSA solution as the blocking solution, which can further improve the accuracy and precision of the detection results. Therefore, the present application selects BSA as the effective component in the blocking solution.

[0145] By comparing the detection results of Examples 1-5, it can be seen that when the concentration of BSA solution in the blocking solution is controlled within the range of 1-1.5 wt%, the accuracy and precision of the detection results can be further improved. Therefore, the present application controls the concentration of BSA solution within the above range.

[0146] By comparing the detection results of Examples 3, 8-11, it can be seen that when the weight ratio of lanthanide ions to anti-rHPV68 L1 protein CD68 monoclonal antibody is controlled within the range of (0.1-0.3):1, the accuracy and precision of the detection results can be further improved. Therefore, the present application controls the weight ratio of lanthanide ions to anti-rHPV68 L1 protein CD68 monoclonal antibody within the above range.

[0147] By comparing the test results of Examples 3, 12-15, it can be seen that when the concentration of the primary antibody is controlled within the range of 2-4 μg / mL, the accuracy and precision of the test results can be further improved. Therefore, the concentration of the primary antibody is controlled within the above range in this application.

[0148] By comparing the test results of Examples 3, 16-19, it can be seen that when the concentration of the secondary antibody is controlled within the range of 180-220 μg / mL, the accuracy and precision of the test results can be further improved. Therefore, the concentration of the secondary antibody is controlled within the above range in this application.

[0149] (2) Sensitivity of the detection method

[0150] Detection method: Using the detection method provided in Example 3, standard solutions of varying concentrations, 0 ng / mL, 10 ng / mL, 50 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL, were added and the fluorescence signal values were detected. A standard curve was constructed using the double logarithm of the concentration versus fluorescence signal values. Ten replicate measurements were performed using a zero reference standard as the sample. The mean fluorescence value, M, and standard deviation, SD, were calculated. The fluorescence value obtained by substituting M + 2SD into the standard curve equation was used, and the calculated concentration value was used as the sensitivity of the detection method.

[0151] Test results: Figure 2 shown. Figure 2 This is the “concentration-fluorescence value” standard curve provided in Example 3.

[0152] Depend on Figure 2 The standard curve shows that the logarithm of the concentration and the logarithm of the fluorescence value are distributed on or around a straight line. The curve equation is: LogY = 0.9874 × LogX + 3.808 (R 2 =0.9972). This standard curve shows that the concentration value of the rHPV68 protein antigen content has a good linear relationship with the fluorescence signal value. Therefore, the detection method provided by this application can quickly obtain the content of the rHPV68 protein antigen in the sample to be tested through this standard curve.

[0153] After calculation, the detection limit of the detection method in this application is 0.07 ng / mL, and the linear range of the fluorescence signal value is 10-1000 ng / mL. The above test results show that the detection method provided by this application has high sensitivity.

[0154] (3) Specificity of the detection method

[0155] Detection method: Using the detection method provided in Example 3 of the present application, high-concentration protein antigens of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59 with a concentration of 20000 ng / mL each were used as test samples to detect the contents of the above 14 protein antigens similar to the HPV68 protein antigen, and the specificity of the detection method was investigated.

[0156] Among them, the cross-reactivity rate = measured concentration / theoretical concentration × 100%.

[0157] Detection results: As shown in Table 6.

[0158] Table 6 Specificity detection results of the detection method

[0159]

[0160]

[0161] Combined with Table 6, by analyzing the specificity detection results, it can be seen that when using the detection method provided in the present application to detect 14 protein antigens similar to the HPV68 protein antigen, the measured concentrations of these 14 protein antigens are far lower than the theoretical concentrations, and the calculated cross-reactivity rates do not exceed 0.0009%. The above detection results indicate that when the detection method provided in the present application is used to detect the HPV68 protein antigen, there is no cross-reaction with other similar protein antigen types and it will not be affected by similar protein antigens. Therefore, the detection method provided in the present application has high specificity.

[0162] (4) Accuracy of the detection method when detecting test samples with different concentrations

[0163] Detection method: According to the preparation method of the test samples in Table 7, samples with different concentrations were prepared as test samples, and the recovery test was carried out using the detection method provided in Example 3 of the present application. Each sample was tested 6 times, and the recovery rate of the test samples was calculated to investigate the accuracy of the detection method.

[0164] Table 7 Preparation method of test samples

[0165]

[0166] Detection results: As shown in Table 8.

[0167] Table 8 Recovery detection results of the detection method

[0168] Sample to be measured Expected concentration (ng / mL) Detected concentration (ng / mL) Recovery rate (%) 1 33.33 35.92 107.77 2 145 147.82 101.94 3 255 246.74 96.76

[0169] Combined with Table 7-8, by analyzing the recovery rate test results of the detection method, it can be seen that when the detection method of the present application is used to detect samples to be measured with different concentrations, the obtained recovery rates are between 96.76% and 107.77%, approaching 100%, indicating that the detection method provided by the present application has high accuracy.

[0170] (5) Precision of the detection method when detecting samples to be measured with different concentrations

[0171] Detection method: Prepare reference products into three samples to be measured with low concentration of 10 ng / mL, medium concentration of 200 ng / mL, and high concentration of 1000 ng / mL respectively. Use the detection method provided in Example 3 of the present application to conduct within-batch and between-batch tests on the above three samples to be measured with different concentrations, calculate the coefficient of variation CV respectively, and examine the precision of the detection method.

[0172] Test results: As shown in Table 9.

[0173] Table 9 Precision test results of the detection method

[0174]

[0175] Combined with Table 9, by analyzing the precision test results of the detection method, it can be seen that when the detection method provided by the present application is used to detect antigens, the coefficient of variation of within-batch test is between 0.73% and 1.18%, and the coefficient of variation of between-batch test is between 1.21% and 2.78%. The coefficients of variation of within-batch and between-batch tests are both less than 3%, indicating that when the detection method provided by the present application is used to detect antigens, it has high precision, is not interfered by other impurities, and has good reproducibility.

[0176] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. An anti-rHPV68 L1 protein CD68 monoclonal antibody, characterized in that, The DNA sequence of the anti-rHPV68 L1 protein CD68 monoclonal antibody is as shown in SEQ ID NO.

1.

2. A TRFIA detection kit for the content of rHPV68 protein antigen, characterized in that, The detection kit includes: the anti-rHPV68 L1 protein CD68 monoclonal antibody described in claim 1 as the primary antibody; the anti-rHPV68 L1 protein CD68 monoclonal antibody labeled with lanthanide ions as the secondary antibody; and the rHPV68 type protein antigen standard sample.

3. The TRFIA detection kit for the content of rHPV68 protein antigen according to claim 2, wherein, The concentration of the primary antibody is 2 - 4 μg / mL.

4. The TRFIA detection kit for the content of rHPV68 protein antigen according to claim 2, wherein The concentration of the secondary antibody is 180 - 220 μg / mL.

5. The TRFIA detection kit for the content of rHPV68 protein antigen according to claim 2, wherein The weight ratio of the lanthanide ions to the anti-rHPV68 L1 protein CD68 monoclonal antibody in the secondary antibody is (0.1 - 0.3):

1.

6. The TRFIA detection kit for the content of rHPV68 protein antigen according to claim 2, wherein The detection kit further includes a blocking solution and an enhancement solution.

7. The TRFIA detection kit for the rHPV68 protein antigen content according to claim 6, wherein The blocking solution is selected from one or more of BSA solution, skim milk powder solution, and horse serum albumin solution.

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