Monoclonal neutralizing antibody for resisting human papilloma virus 31 and application of monoclonal neutralizing antibody
An anti-human papilloma, monoclonal antibody technology, used in antiviral agents, antiviral immunoglobulins, applications, etc.
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Embodiment 1
[0024] Embodiment 1. Hybridoma cell line development and antibody purification of monoclonal antibody:
[0025] 1. Animal immunity
[0026] 1) Basic immunization: HPV31 L1 VLP antigen was mixed with Freund's complete adjuvant in equal volumes and fully emulsified, subcutaneously injected in points, and a total of 6 mice were immunized, and the injection volume of each Balb / c mouse was 10 μg.
[0027] 2) Booster immunization: The emulsion of antigen and Freund's incomplete adjuvant is used for booster immunization. Three days before cell fusion, saline solution containing 15ug of antigen was injected intraperitoneally. The experimental results are shown in Table 1.
[0028] Table 1 Serum detection of immunized mice (OD450)
[0029]
[0030] It can be seen from the above table that the serum titer of No. 5 mouse is the highest, and the spleen of this mouse was selected for fusion with SP20 cells. That is, mouse No. 5 with the strongest signal was selected for fusion and s...
Embodiment 2 19B4 and 19
[0070] Example 2, Identification of 19B4 and 19C2 Antibodies
[0071] 1. Identification of conformational epitopes of antibodies
[0072] Various types of VLP proteins are processed by alkali denaturation and heat denaturation to destroy the secondary or tertiary structure and preserve the primary structure. Then react with monoclonal antibody and detect it by indirect method ELISA. Through this experiment, not only the recognition status of the antibody to various types of VLPs can be identified, but also whether the antibody is a conformational epitope recognition antibody can be identified. If the OD450 value of the protein denatured and reacted with the monoclonal antibody is significantly reduced, it proves that the monoclonal antibody is a conformational epitope recognition antibody. Recognizing antibodies.
[0073] Experimental procedure: VLP denatured protein treatment: 0.2M sodium carbonate, 0.01M DTT, pH 10.6, incubate at room temperature for 30 minutes and boil f...
Embodiment 3
[0082] Example 3. Preparation of HPV31 VLP Detection Reagent Using 19B4 Purified Antibody
[0083] Using the ELISA double antibody sandwich method, the antibody pairing experiment was carried out. In this example, clone 19B4 was used as the coating antibody, HRP-labeled clone 19C2 was used as the detection antibody, and the ELISA detection method was determined. The test results are shown in Table 11.
[0084] Table 11 Preliminary pairing results of kits
[0085]
[0086] Preliminary pairing experiment results show that the signal of the paired combination detected by 19C2-HRP coated with 19B4 is higher, which is more suitable for ELISA detection. Therefore, this pairing was used for further ELISA detection.
[0087] Detection method: Dilute the coating antibody 19B4 with 0.05 mol / L carbonate buffer solution of pH 9.6 to 10 μg / mL, add 100 μL to each well of the microtiter plate, coat at 4°C overnight, pour off the coating solution, Wash twice with PBST, pat dry, then ad...
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