Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof

A technology of antibodies and vectors, applied in applications, antibodies, antiviral agents, etc., can solve the problems of low expression, culture survival, HPV virus failure, etc.

Active Publication Date: 2016-04-27
ATTOGEN BIOMEDICAL SUZHOU INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are three main reasons why there are no suitable antibodies for clinical HPV detection: 1. The expression of HPV protein in clinical tissue or cell samples is low, and high-affinity antibodies are required for detection; 2. The HPV virus is under the existing standard tissue culture technology. Can not survive in laboratory culture; 3, E7 protein itself has immunosuppression, so that the use of E7 protein to immunize animals cannot obtain a good immune response, and the prepared antibodies often cross-react with other HPV proteins. E7 protein does not have specificity

Method used

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  • Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof
  • Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof
  • Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof

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preparation example Construction

[0176] Preparation of monoclonal antibodies

[0177] Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, an antigen of the invention may be administered to an animal to induce the production of monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J. Immunol. 6:511, 1976; Kohler et al., Eur.J.Immunol. 6:292,1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or can be prepared by recombinant DNA methods (US Patent No. 4,816,567).

[0178] Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to culture medium (HAT medium matrix), including myeloma cell lines, such as murine Myeloma cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (avai...

Embodiment 1

[0228] 1. Preparation of human papillomavirus HPV18E7 monoclonal antibody

[0229] 1.1 Animal immunity

[0230] 6-8 weeks old female BALB / C mice were taken, the immunogen was GST-HPV18E7 protein, and the immunization program was shown in Table 1. Before each immunization, blood was collected by tail docking of mice, and Hi s - HPV18E7 was used as the detection antigen to coat the indirect ELISA method to detect the mouse serum titer. Splenocytes were taken for fusion when the serum titer titer of the immunized mice reached the maximum and no longer increased.

[0231] Table 1 Mouse immunization program

[0232]

[0233]

[0234] 1.2 Cell Fusion and Culture

[0235] The splenocytes of immunized mice were collected and fused with myeloma cells SP2 / 0 according to conventional methods. The fusion ratio of splenocytes: SP2 / 0=5:1. Selective culture in CO2 constant temperature incubator. After the fusion, the HAT medium was used to change the medium three times; when the ...

Embodiment 2

[0261] Immunocytochemical staining to detect the specificity of HPV18E7 monoclonal antibody MAB-F2 in suspended and fixed cervical cancer cell lines:

[0262] The cervical cancer cell line Hela cells expressing HPV18E7 protein and the cervical cancer cell line C-33A cells without HPV DNA were fixed with different cell fixatives and then immunocytochemical staining was performed with monoclonal antibody MAB-F2. The specific experimental method is as follows:

[0263] Collect C-33A cells and Hela cells respectively, fix them with 4% paraformaldehyde or LBC fixative for several days, and take a total of 50,000 cells, in which cervical cancer cell Hela cells and negative control C-33A cells are mixed in a certain proportion, Hela The ratio of the number of cells to C-33A cells was 1:9, 1:19, 1:49, 1:99, 1:499, 1:999. After mixing, put it on poly-L-Lysine-treated coverslip and air-dry at room temperature to fix the cells on the coverslip; add TBS washing solution to wash for 5 min...

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Abstract

The present invention provides a monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof. According to the present invention, with the monoclonal antibody, the cervical cancer biomarker HPV18E7 protein in the tumor cells can be detected in a highly specific manner so as to distinguish between the cancerous cervical epithelial cells and the abnormal cervical epithelial cells or non-cancerous cervical epithelial cells and provide basis for the accurate diagnose of the cancer caused by the HPV infection, such that the treatment can be timely taken, the cancer occurrence or cancer spreading can be prevented, and the suffering of patients can be alleviated.

Description

technical field [0001] The invention belongs to the field of biological diagnosis and medicine, in particular, the invention relates to a monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and its application. Background technique [0002] Cervical cancer is a common malignant tumor of the female reproductive system, ranking second among female malignant tumors. In 1949, Sttauss first observed human papillomavirus (humanpapillomavirus, HPV) particles in wart leachate under an electron microscope. After Harald ZurHausen proposed that HPV may be a sexually transmitted carcinogen in 1976, the research on the relationship between HPV infection and cervical cancer has become a hot topic in the study of tumor virus etiology. A large number of studies have found that HPV is the culprit of cervical cancer and can also cause a variety of other tumors, including reproductive tract, breast, digestive tract and respiratory tract cancers. In recent year...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13C12P21/02A61K39/395A61P31/20A61P35/00G01N33/571G01N33/574
Inventor 常小迦施丽君时成龙韩凤丽
Owner ATTOGEN BIOMEDICAL SUZHOU INC
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