Monoclonal antibody for recognizing high-risk hpv E7 protein and its application

An antibody and protein technology, applied in the direction of antibodies, applications, antiviral immunoglobulins, etc., can solve the problems of heavy workload of pathologists, restricting the efficiency and coverage of early diagnosis of cervical cancer, etc.

Active Publication Date: 2021-05-28
ATTOGEN BIOMEDICAL SUZHOU INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cytological interpretation requires morphological evidence, which is unavoidable subjectivity and causes extremely heavy workload for pathologists
These factors have greatly restricted the efficiency and coverage of early diagnosis and individualized treatment of cervical cancer in my country.

Method used

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  • Monoclonal antibody for recognizing high-risk hpv E7 protein and its application
  • Monoclonal antibody for recognizing high-risk hpv E7 protein and its application
  • Monoclonal antibody for recognizing high-risk hpv E7 protein and its application

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0171] Preparation of monoclonal antibodies

[0172] 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. For monoclonal antibodies, hybridoma technology can be used to prepare (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 available recombinant DNA method (US Patent No. 4,816,567).

[0173]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...

Embodiment 1

[0208] 1. Preparation of rabbit monoclonal antibody against human papillomavirus HPV16E7

[0209] 1.1 Screening of single-chain antibody (scFv)

[0210] 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 (t...

Embodiment 2

[0228] Example 2 Using immunocytochemical staining to detect the expression of HPV16E7 protein in tumor cells fixed by liquid-based fixative

[0229] The CaSki and C-33A cells were collected by centrifugation in PBS, centrifuged to remove excess PBS, and then fixed with TCT fixative for 30 min. The fixed CaSki and C-33A cells were smeared on glass slides, soaked in 95% ethanol for 30 minutes, and air-dried overnight.

[0230] After the air-dried tumor cell coverslips were placed in 50% ethanol for 10 min, they were transferred to deionized water for at least 30 s. To prevent non-specific background staining, do not allow coverslips to dry out during staining. Place the deionized water-treated cell slides in Tris-EDTA (pH 9.0) repair solution, heat repair at 95-99°C for 10 minutes, rewarm at room temperature together with the repair solution for 20 minutes; spin dry, add PBST washing solution to wash 5min; shake dry, add 3% H to inactivate endogenous peroxidase 2 o 2 / PBS a...

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Abstract

The present invention provides a monoclonal antibody for identifying high-risk HPV E7-positive cervical epithelial cancer cells and its application. The antibody can detect cervical cancer biomarker HPV E7 protein in tumor cells with high specificity, covering high-risk HPV16, HPV31, HPV35, High-risk subtypes such as HPV52 and HPV58 can distinguish cancerous cervical epithelial cells from cervical abnormalities or non-cancerous cervical epithelial cells, providing evidence for doctors to accurately diagnose cancers caused by HPV infection, and can effectively reduce the missed diagnosis rate of high-grade cervical lesions , provide sufficient time and basis for clinicians to diagnose and treat patients, and improve early detection and early intervention of cervical diseases. At the same time, unnecessary colposcopy can also be reduced and avoided.

Description

technical field [0001] The invention belongs to the field of biological diagnosis and medicine. Specifically, the invention relates to a monoclonal antibody for identifying human papillomavirus (HPV) subtype 16 positive tumor cells, including human cervical epithelial cancer cells, and its application. Background technique [0002] Cervical cancer is the second most common female malignancy. About 500,000 women are diagnosed with cervical cancer every year in the world, and more than half of them die from it. The early symptoms of cervical cancer are not obvious, and there is a long and reversible precancerous lesion period in the development process. According to statistics, about 20% of low-grade cervical injuries will turn into high-grade injuries, and if not treated in time, 30% of them will further turn into malignant tumors. It takes about 10 years for the general cervical precancerous lesions to develop into cervical cancer. During this period, early diagnosis of pre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08C12N15/13A61K39/395A61P31/20A61P35/00G01N33/68G01N33/574G01N33/569
Inventor 常小迦刘岩时成龙韩凤丽施丽君
Owner ATTOGEN BIOMEDICAL SUZHOU INC
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