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Monoclonal neutralizing antibody of HPV52L1 and application of monoclonal neutralizing antibody

A technology of monoclonal antibody, cgmccno.19186, applied in applications, antibodies, antiviral agents, etc., can solve the problems of limiting monoclonal antibody detection of HPV52 virus HPV52 virus treatment, etc., and achieve good sensitivity and specificity

Active Publication Date: 2020-10-02
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

It can be seen from the above data that HPV52 is a common high-risk type next to HPV16, but there are very few studies on its monoclonal antibody, which limits the use of monoclonal antibodies to detect HPV52 virus and treat HPV52 virus

Method used

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  • Monoclonal neutralizing antibody of HPV52L1 and application of monoclonal neutralizing antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Embodiment 1, establishment of hybridoma cells

[0097] 1. Animal immunization

[0098] Using the Escherichia coli expression system, the virus packaging particles of the L1 protein of HPV52 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 1:16000, >1:32000, and 1:16000 respectively, and the No. 2 mouse with the highest titer was selected for fusion .

[0099] The operation steps of the indirect E...

Embodiment 2

[0108] Example 2: Preparation and identification of monoclonal antibodies

[0109] 1. Preparation of monoclonal antibody against HPV52 from mouse ascites

[0110] Adult BALB / c mice were selected, and hybridoma cells 52-1F5 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.

[0111] 2. Antibody Purity Determination

[0112] The purified antibody was subjected to 12% SDS-PAGE electrophoresis, and the results showed that the purity was above 95%...

Embodiment 3

[0120] Example 3: Detection of antibody neutralization activity

[0121] Through the pseudovirus-cell neutralization model, the neutralizing activity of the antibody was tested.

[0122]First dilute the antibody with PBS to 1000ng / mL, 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, 35, 39, 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...

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Abstract

The invention relates to the technical field of antibody drugs, in particular to a monoclonal neutralizing antibody of HPV52L1 and an application thereof. The invention provides a specific monoclonalneutralizing antibody of HPV52 and a hybridoma cell line of generating the antibody. One monoclonal neutralizing antibody with the highest neutralization activity titer can be widely applied to detection of HPV52 virus infection in clinical samples by using an indirect ELISA method, a double-antibody sandwich ELISA method, an immunological hybridization method and the like; and the monoclonal neutralizing antibody with the highest neutralization activity titer can be used to prepare a vaginal gel, a vaginal cleaning fluid, a topical lotion, a vaginal suppository, freeze-dried powder, a vaginaleffervescent tablet and the like, is applied to treatment of HPV52 infection patients and can effectively improve the clinical negative conversion rate of the HPV52 patients. The monoclonal neutralizing antibody is of great significance to the healthy development of women and public health prevention and control.

Description

technical field [0001] The invention relates to the technical field of antibody medicine, in particular to a monoclonal neutralizing antibody of HPV52L1 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, among which HPV16 and HPV18 are the most common carcinogenic types, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13G01N33/569A61K39/42A61P31/20A61P35/00A61P17/12
CPCC07K16/084G01N33/56983A61P31/20A61P35/00A61P17/12C07K2317/76C07K2317/56A61K2039/505G01N2333/025
Inventor 魏颖颖郭光华蔺皓邹国宝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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