[Identification and characterization of monoclonal antibody Y4F11 against human B7-H3].
Objective To generate mouse anti-human monoclonal antibodies against the molecules expressed on immune cells from malignant pleural effusions, and identify the antigen recognized by the monoclonal antibody named Y4F11. Methods The cells separated from pleural effusions of lung cancer patients were used to immunize BALB/c mice. Hybridoma technology was used for cell fusion and screening; the monoclonal antibody named Y4F11 was chosen as the object for the following experiment. Flow cytometry was performed to analyze the molecules expressed on naive and activated T, B, NK cells and monocytes; Co-immunoprecipitation was conducted to get the antigen pulled down by Y4F11. Mass spectrometry sequencing and BLASTP were used to identify the antigen molecules. Western blotting, dot-blotting and flow cytometry were utilized to identify Y4F11. Results The study obtained 78 hybridoma clones secreting antibodies stably which could recognize surface molecules on cells from pleural effusions. The hybridoma named Y4F11 was chosen as the following experiment object for further identification. The molecule which Y4F11 recognized was about 50 Kd, and further was confirmed as B7-H3 molecule. Furthermore, Y4F11 could recognize B7-H3 expressed on B7-H3 gene-transferred cells as well as B7-H3 fusion protein. Besides, the expression pattern of B7-H3 on immune cells was confirmed by Y4F11 antibody. All these results indicated that the antigen molecule recognized by Y4F11 antibody was B7-H3. Conclusion A hybridoma cell line with stable expression of monoclonal antibody B7-H3 has been successfully obtained and could be used for biological function study on B7-H3.
College of Biology and Basic Medical Science, Soochow University, Suzhou 215000, China.
Zhengpeng
Z
Shi
College of Biology and Basic Medical Science, Soochow University, Suzhou 215000, China.
Yumin
Y
Hu
College of Biology and Basic Medical Science, Soochow University, Suzhou 215000, China.
Hanqing
H
Chen
Jiangsu Provincial Institute of Clinical Immunology, First Affiliated Hospital, Soochow University, Suzhou 215000, ChinaJiangsu Provincial Institute of Clinical Immunology, First Affiliated Hospital, Soochow University, Suzhou 215000, China. *Corresponding authors, E-mail: xueguangzh@126.com.
Fengqing
F
Fu
Jiangsu Provincial Institute of Clinical Immunology, First Affiliated Hospital, Soochow University, Jiangsu Provincial Key Laboratory of Stem Cell and Biomedical Material, Soochow University, Suzhou 215000, China. *Corresponding authors, E-mail: fufengqing@suda.edu.cn.
Xueguang
X
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D060887:B7 Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000276:immunology; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D060887:B7 Antigens; C423237:CD276 protein, human