A Novel Protocol for Making of Monoclonal Antibody against AFP

Abstract

Abstract-Alpha-fetoprotein (AFP) is a major serum protein (Mr, =70,000) synthesized during fetal life. Reappearance of AFP in adult serum often signals pathological conditions, particularly hepatocarcinomas and teratocarcinomas. To obtain monoclonal antibodies against AFP for further study of the structure and biological function of AFP, a novel method for making hybridoma cell lines producing monoclonal antibodies for AFP by immunization of animals with synthetic peptides were proposed. One epitope peptide was synthesized based on the bioinformatics analysis of the AFP and coupled with keyhole limpet hemocyanin ( KLH ). BALB/c mice were immunized with the coupled AFP polypeptide to make monoclonal antibody. Hybridomas were generated by the fusion of the spleenocytes from these mice with Sp2/0 myeloma cells. Hybridomas producing anti-AFP antibodies were screened by enzyme-linked immunosorbent assay ( ELISA), and the monoclonal antibodie specificities were determined by and indirect-ELISA.The favorite epitope of AFP was seleceted by bioinformatics methods and synthesized successfully, which coupled with carrier KLH as immunogen to immunize BALB/c mice, two hybridoma cell lines (2F12 and 3G8) stable in secreting anti-AFP specificity monoclonal antibodies (mAbs) were successfully generated, Titers of tybridoma cell culture supernatant and asicites are both above 1:32 and 100,000 respectively.Creatively preparing mAb against AFP by ways of immunizing mice with the coupled epitope to KLH.The mAb should be suitable for studies of the cellular localization of AFP's biosynthesis and its processing after interaction with receptor. It will also be useful in the immunoaffinity purification of the AFP in sreum and as a useful reagent for the analysis of biochemical, structural, and functional properties of AFP