Monoclonal and polyclonal antibodies against the precursor of recombinant human insulin

• Published in: Acta biochimica et biophysica Sinica 2013-08, Vol.45 (8), p.704-705
• Main Authors: Leng, Chunsheng, Li, Qingwei, Wu, Fenfang, Chen, Liyong, Su, Peng
• Format: Article
• Language: English
• Subjects: Antibodies - immunology; Antibodies, Monoclonal - immunology; Humans; Insulin - immunology; Protein Precursors - immunology; Recombinant Proteins - immunology; Sepharose; Sigma公司; 前体; 单克隆抗体; 多克隆抗体; 重组人生长激素; 重组人胰岛素; 间接ELISA法
• ISSN: 1672-9145; 1745-7270
• DOI: 10.1093/abbs/gmt041

The precursor of recombinant human insulin is also called recombinant proinsulin. It is a fusion protein containing pro tection peptide and insulin. The protection peptide contains 55 amino acids （48 amino acids from human growth hormone, and a connecting peptide containing Argresidues） and can effectively improve protein stability and expression efficiency [1]. However, in clinical trials the presence of pro insulin in the insulin final products was found to have a negative effect on the heart, leading to an increased inci dence of myocardial infarction [26]. More importantly, re combinant proinsulin is a heterologous protein. Therefore, the removal of the recombinant proinsulin in the final product of insulin is vital, and the effectiveness of its removal should be analyzed by appropriate methods. In this study, we prepared specific monoclonal and polyclonal anti bodies against proinsulin and established enzymelinked im munosorbent assay （ELISA） for the detection of the residual precursor in the insulin production. Three adult female BALB/c mice were intraperitoneally injected with 55peptide as an antigen. Each mouse was injected with 100 pog antigen mixed with complete Freund＇s adjuvant （Sigma, St Louis, USA） the first time, and then boosted with 50 Ixg antigen mixed with incomplete Freund＇s adjuvant for three times. The injection interval was 2 weeks. After four immunizations, spleen cells were fused with SP2/0 myeloma cells and the hybridoma cells were selected by growing in HAT （hypoxanthine, aminopterin, and thymidine） medium. Monoclonal cell lines were screened by the limiting dilution method. To obtain a large amount of antibodies, the hybridoma cells were injected into the peritoneal cavity of mice, and antibodyrich ascites was collected. The antibodies were purified by proteinG Sepharose （GE Healthcare, Wisconsin, USA）. The titer of anti55peptide monoclonal antibody （mAb） was deter mined by indirect ELISA. New Zealand white rabbits were immunized with the purified recombinant proinsulin （but not insulin）. If theanimal is immunized with pure insulin, it would soon become sick and die. Each rabbit was hypodermically injected with 600μg antigen mixed with complete Freund＇s adjuvant the first time, and then boosted three times with 300 μg antigen mixed with incomplete Freund＇s adjuvant. The injection interval was 2 weeks. Seven days after the last immunization, the serum of the immunized animals was col lected. The antiinsulin polydonal antibodies （