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Anti-HPV31L1 monoclonal neutralizing antibodies and application thereof

A monoclonal antibody and sequence technology, applied in applications, antibodies, antiviral agents, etc., can solve the problems of less research on monoclonal antibodies and limit the detection of HPV31 virus by monoclonal antibodies, and achieve good sensitivity and specificity.

Active Publication Date: 2020-05-29
中生方政生物技术股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above data show that HPV31 is one of the common HPV types in patients with cervical cancer, no matter from the rate of HPV infection or the proportion of cervical lesions, but there are very few studies on its monoclonal antibody, which limits the use of monoclonal antibodies Antibody detection of HPV31 virus, and treatment of HPV31 virus

Method used

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  • Anti-HPV31L1 monoclonal neutralizing antibodies and application thereof

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 HPV31 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 anti-HPV31L1 monoclonal antibody from mouse ascites

[0111] Adult BALB / c mice were selected, and hybridoma cells 31-2G7 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] The neutralizing activity of the antibody was tested by a pseudovirus-cell neutralization model.

[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. Then observe the fluorescence situation, collect the cells and detect the fluorescence by flow cytometry, if there is an inhibitory effect, calculate the fluorescence inhibition rate according to the fluorescence inhibition rate=(1-experimental group / control gro...

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Abstract

The invention relates to the technical field of antibody drugs, in particular to anti-HPV31L1 monoclonal neutralizing antibodies and application thereof. The invention provides the specific monoclonalneutralizing antibodies of HPV31 and a hybridoma cell line for producing the antibodies. One monoclonal neutralizing antibody with the highest neutralizing activity titer in the monoclonal neutralizing antibodies can be widely applied to infection detection of the HPV31 virus in clinical samples in virtue of an indirect ELISA method, a double-antibody sandwich ELISA method, an immune hybridization method and the like; the monoclonal neutralizing antibody with the highest neutralizing activity titer is used for preparing a vaginal gel, a vaginal cleaning fluid, an external lotion, a vaginal suppository, freeze-dried powder, a vaginal effervescent tablet and the like for treating HPV31 infected patients, and can effectively improve the clinical negative conversion rate of the HPV31 patients. The anti-HPV31L1 monoclonal neutralizing antibodies and the application thereof in the invention are of great significance to the healthy development of women and the prevention and control of public health.

Description

technical field [0001] The invention relates to the technical field of antibody medicines, in particular to monoclonal neutralizing antibodies against HPV31L1 and applications thereof. 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 typ...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13G01N33/577G01N33/569A61K39/42A61P35/00A61P17/12A61P31/20
CPCC07K16/084G01N33/577G01N33/56983A61P35/00A61P17/12A61P31/20C07K2317/76C07K2317/565G01N2333/025A61K2039/505
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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