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Monoclonal antibody for detection and classification of cervical cancer and application thereof

A technology of antibody and detection plate, which is applied in the field of biological diagnosis and medicine, and can solve the problems of culture survival, failure of HPV virus, failure to obtain immune response, etc.

Active Publication Date: 2015-12-09
ATTOGEN BIOMEDICAL SUZHOU INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently no suitable antibodies for clinical HPVE7 detection. There are two main reasons: 1. The expression of HPV protein in clinical tissue or cell samples is low, and high-affinity antibodies are needed for detection; 3. The E7 protein itself has immunosuppression, so that the animals immunized with E7 protein cannot obtain a good immune response

Method used

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  • Monoclonal antibody for detection and classification of cervical cancer and application thereof
  • Monoclonal antibody for detection and classification of cervical cancer and application thereof
  • Monoclonal antibody for detection and classification of cervical cancer and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0181] Preparation of monoclonal antibodies

[0182] 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), phage display technology or recombinant DNA method (US Patent No. 4,816,567).

[0183]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...

Embodiment 1

[0215] 1. Preparation of rabbit monoclonal antibody against human papillomavirus HPV16E7

[0216] 1.1 Screening of single-chain antibody (scFv)

[0217]Rabbits were immunized with His-HPV16E7 recombinant protein, and the titer was detected with His-HPV16E7 recombinant protein and His irrelevant protein. Isolation of rabbit B lymphocytes to obtain immunoglobulin genes. The complete set of variable region genes of B cells is cloned and assembled into a phage antibody library. The constructed phage antibody library was panned with the recombinant protein His-HPV16E7. Enrichment after three rounds of panning; determination of phage titer; amplification of phage plaques; DNA sequencing; Among them, the recombinant protein His-HPV16E7 was selected for ELISA detection and screening, and the negative control (N) was set with His irrelevant protein, the Anti-6×His antibody was set as the positive control (P) coated with His antigen, and the blank control was set at the same time (th...

Embodiment 2

[0235] Example 2 Rabbit-derived monoclonal antibody RAB-034 is used to detect cervical lesion tissue by immunohistochemical staining

[0236] According to the results of antibody screening, rabbit monoclonal antibody RAB-034 was used as the primary antibody to further explore its application in the detection of cervical precancerous lesions. Cervical intraepithelial neoplasia (CIN) is the precancerous lesion of cervical cancer, including cervical mild (CINI grade), moderate (CINII grade), severe dysplasia and carcinoma in situ (CINIII grade). Select normal cervical tissue paraffin sections (negative control), CINI, -II, -III pathological paraffin sections and cervical cancer paraffin sections (positive control), and use RAB-034 to carry out immunohistochemical staining detection, specific operations refer to Example 1, 2.4 section. Results: There were 7 samples of CINI grade, 3 of which had specific staining, and the positive rate was 42.9%; 16 samples of CINII-III grade, 10 ...

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Abstract

The invention provides a monoclonal rabbit antibody for identifying HPV16 positive cervical tissues. The antibody can be used for specifically detecting a biomarker HPV16E7 protein in tissues including cervical cancer and cervical lesions, so that HPV persistent infection related cervical cancer tissues from abnormal or noncancerous cervical epithelial tissues, cervical cancer caused by high-risk HPV infection can be accurately diagnosed, and risk early-warning can be especially performed for whether early cervical lesions cancerate or not, so that the misdiagnosed rate of cervical lesions can be effectively reduced, and injuries and resource waste caused by over treatment for patients can be improved.

Description

technical field [0001] The invention belongs to the field of biological diagnosis and medicine, in particular, the invention relates to the identification and early risk warning of HPV16E7 protein in cervical cancer and cervical lesion tissue. Background technique [0002] Cervical cancer is a common malignant tumor of the female reproductive system, ranking second among female malignant tumors. The 5-year survival rate of advanced cancer is low, and it has high morbidity and mortality worldwide. In 1976, zur Hansen proposed that human papillomavirus (HPV) may be a sexually transmitted carcinogen, and began to study the relationship between HPV and cervical cancer. At present, many epidemiologists have confirmed 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. zur Hansen also won the Nobel Prize in Physiology or Medicine in 2008. In recent years, th...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/63A61K39/395A61P35/00G01N33/68G01N33/574
Inventor 常小迦时成龙韩凤丽施丽君
Owner ATTOGEN BIOMEDICAL SUZHOU INC
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