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Preparation of a Monoclonal Antibody Against Human 4IgB7-H3 Molecule and Its Primary Application

Author: HeNa
Tutor: ZhouYingHui；ZhangXueGuang
School: Suzhou University
Course: Biochemistry and Molecular Biology
Keywords: 4IgB7-H3 2IgB7-H3 Monoclonal antibodies Expression Analysis Glioma
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 24
Quote: 0
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Abstract


Costimulatory molecules (co-stimulatory molecules) plays an important role in immune regulation, B7-H3 is a newly discovered synergistic stimulation members of the family of molecules. B7-H3 shear two different forms: B7-H3a and B7-H3b. The B7-H3a ectodomain IgV-IgC two immunoglobulin domains, is also called 2IgB7-H3, B7-H3b ectodomain IgV1-IgC1-IgV2-IgC2 four immunoglobulin domains it is also known 4IgB7-H3. Extremely wide range of expression levels of B7-H3mRNA, in almost all of the lymphoid tissue and non-lymphoid tissues could be detected mRNA transcription of the molecule, which the expression of 4IgB7-H3mRNA abundance is far higher than 2IgB7 H3mRNA Therefore, previous studies that 4IgB7- H3 is the main form of expression of B7-H3. But because there is no specific recognition 4IgB7-H3 monoclonal antibody, it is not clear 4IgB7-H3 protein expression profiles in the organization. In this study, prepared one can specifically recognize 4IgB7-H3 murine anti-human B7-H3 monoclonal antibody, followed by research and analysis of the molecular expression profiling. Objective: To develop specific recognition of mouse anti-human 4IgB7-H3 monoclonal antibody and its preliminary application. Method: high expression 4IgB7-H3 293T cells as immunogens, the routine immunization mice, cell fusion and screening. L929/4IgB7-H3 gene transfected cells for antibody screening positive cells, transfected cells L929/2IgB7-H3 gene screening for antibody-negative cells, repeated screening of hybridoma cells by indirect immunofluorescence and flow cytometry, and multiple subcloned, Flow cytometry analysis of the antibody on the identification of the different members of the B7 family of gene transfected cells. Loci competition experiments, the recognition specificity of the antibody epitope were identified. Using two antibodies detected by flow cytometry 4IgB7-H3 expression characteristics of the tumor cell lines and immune cells and immune histochemical method to detect two antibodies identified in tumor cell lines and tumor tissues. Results: successfully prepared two continuous hybridoma cell lines stably secreting mouse anti-human B7-H3 monoclonal antibody 4C3 and 9C3, which 2IgB7-H3 monoclonal antibody 4C3 can not only identify but also identify 4IgB7-H3 molecule, while the monoclonal antibody 9C3 only recognizes 4IgB7-H3. The protein level 4IgB7-H3 considerable limitations expressed in some tumor cell lines, human peripheral blood mononuclear cells, and mature dendritic cells; widely expressed B7-H3 (2IgB7-H3 and / or 4IgB7-H3) molecule in A variety of tumor cell lines, monocytes, and dendritic cells. In addition, immunohistochemical analysis showed that 4IgB7-H3-specific expression in glioma tissues, and incrementally with increasing pathological grades of glioma. Conclusion: The hybridoma cell lines obtained two the stable secretion mouse anti-human B7-H3 monoclonal antibody 4C3 and 9C3. 4IgB7-H3 monoclonal antibody 9C3 successful expression of B7-H3 two splicing for further analysis characteristics and biological function provides a material basis.

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