Development and identification of mouse anti-human S100 A9 natural protein / 中国免疫学杂志

ABSTRACT

Objective:To prepare and identify the mouse anti-human monoclonal antibodies ( mAbs) using leukocytes as im-munogens. Methods: The mice were immunized using human peripheral blood leukocytes. Then, use of B lymphocyte hybridoma technology preparation of mAbs,followed screening by immunocytochemistry and limited dilution. The secreted mAbs were identified by immunoprecipitation,mass spectrometry,Western blot,ELISA and immunohistochemistry. Results:The 35 positive polyclonal cells were obtained,of which 11 strains secreted mAbs against S100A9. And one strain was used to prepare monoclonal antibody. The purified mAb against S100A9 were purified and identified as IgG1 subtype,with the titer,purity and affinity constant was 1∶3. 18×105,95% and 3. 54×108 L/mol,respectively. This mAb generally had 0. 12% crossed reactivity to S100A8 ,and showed little or no cross reactivity to S100A12 and S100A13. The prepared monoclonal antibodies can specifically recognizes the S100A9 antigen in human breast cancer tissues. Conclusion:Successful preparation of mAb against S100A9,which can secrete specific mAb against S100A9 protein with high titers and specificity have been established successfully,which laid the foundation for the immunology application.