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Monoclonal neutralizing antibodies of HPV33L1 (human papillomavirus 33L1) and application of monoclonal neutralizing antibodies

A monoclonal antibody, cgmccno.19190 technology, applied in the direction of antibodies, antiviral agents, antitumor drugs, etc., can solve the problems of less research on monoclonal antibodies and limit the detection of HPV33 virus by monoclonal antibodies, and achieve good sensitivity and specificity Effect

Active Publication Date: 2020-05-08
中生方政生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above data show that HPV33 is a common HPV type in cervical cancer patients, both abroad and domestically, but there are very few studies on its monoclonal antibody, which limits the use of monoclonal antibody to detect HPV33 virus and treat HPV33 virus

Method used

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  • Monoclonal neutralizing antibodies of HPV33L1 (human papillomavirus 33L1) and application of monoclonal neutralizing antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Embodiment 1, establishment of hybridoma cells

[0098] 1. Animal immunization

[0099] Using the Escherichia coli expression system, the virus packaging particles of the L1 protein of HPV33 type were prepared as the antigen. For the first immunization, the antigen was mixed with Freund's complete adjuvant in equal volumes and fully emulsified, and injected subcutaneously in points. Each Balb / c mouse The injection volume was 100 μg, and 3 mice were immunized; on the 7th, 14th, and 21st days of the first immunization, the emulsion of antigen and Freund's incomplete adjuvant was used for booster immunization, and on the 14th day, small Blood was collected from the tail vein of the rat, the serum was separated, and the antibody titer was detected by indirect ELISA method. The antibody titer of the mouse serum was >1:32000, 1:8000, 1:16000 respectively, and the No. 1 mouse with the highest titer was selected for fusion .

[0100] The operation steps of the indirect ELISA ...

Embodiment 2

[0109] Example 2: Preparation and identification of monoclonal antibodies

[0110] 1. Preparation of monoclonal antibody against HPV33 from mouse ascites

[0111] Adult BALB / c mice were selected, and hybridoma cells 33-2B6 were inoculated intraperitoneally, 1×10 per mouse 6 -2×10 6 First, when the abdomen is obviously enlarged and the skin feels tense when touched with hands, ascites can be collected with a 16-gauge needle. Centrifuge the ascites (13000r / min for 30 minutes), remove cell components and other precipitates, collect the supernatant, purify the antibody with a ProteinG affinity column, and detect the titer of the ascites and the purified antibody by indirect ELISA. The results show that the ascites was diluted 100,000 times positive, the purified antibody diluted to 1ng / mL is still positive.

[0112] 2. Antibody Purity Determination

[0113] The purified antibody was subjected to 12% SDS-PAGE electrophoresis, and the results showed that the purity was above 95%...

Embodiment 3

[0119] Example 3: Detection of antibody neutralization activity

[0120] Through the pseudovirus-cell neutralization model, the neutralizing activity of the antibody was tested.

[0121]First dilute the antibody to 1000ng / mL with PBS, then dilute the antibody to 0.06ng / mL by 2-fold gradient, take 50μL of each concentration of antibody and 50μL of appropriate concentration of HPV6, 11, 16, 18, 31, 33, 45, 52, 58, 59, and 68 pseudoviruses were incubated in a 96-well plate at 4°C for one hour, and a negative serum control, a positive serum control, a cell control, and a pseudovirus control were set. Then each mixture was added to the 96-well plate pre-coated with 293FT cells and cultured in a cell culture incubator for 72 hours. Afterwards, the fluorescence was observed, and the cells were collected to detect the fluorescence by flow cytometry. If there was an inhibitory effect, the fluorescence inhibition rate was calculated according to the fluorescence inhibition rate = (1-ex...

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Abstract

The invention relates to the technical field of antibody medicines and in particular relates to monoclonal neutralizing antibodies of HPV33L1 (human papillomavirus 33L1) and application of the monoclonal neutralizing antibodies. The invention provides specific monoclonal neutralizing antibodies of HPV33L1 and a hybridoma cell line for generating the antibodies. One monoclonal neutralizing antibody, with the highest neutralization activity titer, of the antibodies, can be widely applied to infection detection on HPV33 viruses in clinical samples by using an indirect ELISA (enzyme linked immunosorbent assay) method, a double-antibody sandwich ELISA method, an immunohybridization method, and the like. One monoclonal neutralizing antibody, with the highest neutralization activity titer, of theantibodies, is prepared into a vagina gel, a vagina cleaning fluid, an external lotion, a vaginal suppository, freeze-dried powder, vagina effervescent tablets, and the like, can be adopted to treatHPV33 infection patients and is capable of effectively increasing the clinical negative conversion ratio of the HPV33 patients. The antibodies have great significances for health development of females and public health control.

Description

technical field [0001] The invention relates to the technical field of antibody medicine, in particular to a monoclonal neutralizing antibody of HPV33L1 and its application. Background technique [0002] Human papillomavirus (HPV) is a spherical, double-stranded DNA virus with a diameter of 55-60nm and a nucleocapsid with 20-hedral symmetry. Capsid protein L2 composition. It mainly invades human epithelial tissues and cells, can cause squamous epithelial proliferation of human skin and mucous membranes, and induce various benign and malignant hyperplastic lesions. High-risk HPV infection is associated with the occurrence of various malignant tumors, while low-risk HPV infection can cause anal and genital warts. [0003] High-risk HPV types include 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82, etc., which can cause cervical intraepithelial neoplasia ( CIN) and cervical cancer, wherein HPV16 and HPV18 are the most common carcinogenic types, and the...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/113C12N5/20A61K39/395A61P31/20A61P17/12A61P35/00
CPCC07K16/084A61P31/20A61P17/12A61P35/00A61K2039/505C07K2317/76C07K2317/565
Inventor 邹国宝魏颖颖郭光华蔺皓刘欣欣宋高尚沈江卫
Owner 中生方政生物技术股份有限公司
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