Preparation Of Anti-human NGF Monoclonal Antibody And Study Of Its Biological Characteristics

[Objective] The monoclonal antibody (mAb) against human NGF and its biological characteristics were studied in order to provide the research basis for clinical prevention and treatment. [Methods] The female Balb/c mice of 6~8 weeks were immunized with NGF obtained from human placenta through hypoderm and peritoneal cavity. The immunized spleencytes were fused with mouse myeloma cells (sp2/0) by polyethylene glycol (PEG). Specific mAbs secreted by hybridomas were screened with Enzyme Linked Immunosorbent Assay (ELISA). Positive hybridomas were repeatedly cloned and screened of specific mAbs. The chromosome number of hybridomas was analyzed. The subclass and type of mAbs were identified with fast-strip analysis. The mAbs were produced by in-vivo procedures in mouse peritoneal cavity and purified by Protein G. The PC 12 cells were cultivated with mAbs to observe PC 12 cells' growth. [Results] Two hybridoma cell lines (1F6 and 5E4) secreting specific anti-NGF mAbs were obtained. Their biological characteristics were that (1) Karyomorphism Analysis of chromosome indicated that the chromosome number was over 100, which showed that 1F6 and 5E4 were hybridomas; (2)Fast-strip method analyses displayed that both mAbs belonged to mouse IgG1 and K ; (3)Two mAbs were produced by in-vivo procedures in mouse peritoneal cavity and the positivity of ascites was 90%. Every mouse could at least produce 8.0 ml ascites; (4)The quantity of ascitic mAb in 1F6 and 5E4 was 2.0mg/ml and 1.5mg/ml respectively; (5)The ascitic liter was over 1:1000 by ELISA; (6)Two hybridomas grew well after long-term culture in vitro and secreted mAbs steadily; (7)The experiment showed that 1F6 and 5E4 mAbs could inhibit NGF of promoting the branches growth in PC 12 cells. [Conclusion] Two hybridoma cells (1F6, 5E4) were obtained secreting specific anti-NGF mAbs continuously and steadily. The important methods of studying the biological functions of NGF were developed. It was a key question to improve the immune quantity, to adjust the immune way of antigen, to keep the activity of sp2/0 cells, to stabilize the temperature of fusion and to enhance the production of ascitic mAb. The results showed 1F6 and 5E4 mAbs could decrease the biological function of NGF keeping the growth of branches of PC 12 cells. NGF had a wide biological rule in nervous system and non-nervous systems, and preparation of anti-human NGF monoclonal antibody had significant clinical values. But anti-NGF monoclonal antibody and its biological activities were obtained only in this paper. How to apply to diagnosis, treatment and prevention clinically, many research works were needed to do.