Isolations And Identifications Of Daintain/AIF-1 And The Monoclonal Antibodies Against Daintain/AIF-1

Daintain/allograft inflammatory factor 1 (AIF-1) is a 146-aa residue hormone-like cytokine discovered recently that is encoded within the HLA class III genomic region with a mass of 17kDa and an acetylated N terminus. Daintain/AIF-1 ,mainly expressed by activated macrophages and T lymphocytes,is involved in Transplant rejection, autoimmune disease and tumor progress etc.For further investigation of this peptide distribution and functions, Daintain/AIF-1 was isolated from porcine intestine with acetic acid extraction ,alginic acid adsorption, and sodium chloride saltingout followed by sephadex G-25(fine)column chromatography,DEAE anion exchange column chromatography and reverse-phase HPLC purification, The molecular mass of the fraction determined by MALDI-TOF-MS was 16.692kDa. After hydrolysised with trypsogen, the fragment separated by RP-HPLC was selected for sequence analysis as YSSDEDLSRK consensus with the 36-45 aa residue of Daintain/AIF-1, So the fraction identifed was Daintain/AIF-1.Daintain/AIF-1 purified was then used to immunize BALB/c mice as the immunogen. Furthermore, Both hybridization technique and enzyme linked immunosorbent assay(ELISA) were performed to generate cell lines which produced monoclonal antibodies(Mabs) against Daintain/AIF-1. Isotypes, titer and affinity of the Mabs belong to G1 subclass withκlight chain. Thereafter, the specificity of monoclonal antibodies was identified by Western blot and competitive inhibition ELISA.The Mabs against Daintain/AIF-1 prepared and identified in first time are useful tools for further investigation of transplant rejection, autoimmune diseases and cancer diagnosis.