Preparation and characterization of respective monoclonal antibodies to KEYHOLE LIMPET HEMOCYANIN and E. coli. ribose binding protein

Abstract

By using an radioimmune assay (RIA), two hybridoma lines that secrete monoclonal antibodies were selected after fusion of mouse $sp^2$/o myeloma cells with spleen cells isolated from Balb/c mice that have been immunized with kyehole limpet hemocyanin (KLH), one of molluscan hemocyanin. They were identified to secrete Ig Gl by double gel immunodiffusion and have no affinity to one of arthropod hemocyanin, portunus trituberculatus hemocyanin, by RIA. Enzyme linked immunosorvent assay (ELISA) was compared with RIA and showed that ELISA is more sensitive than RIA at least in our laboratory condition. E. coli ribose binding protein (RBP) was purified by one step DEAE-Sephadex chromatography and by using ELISA 6 positive wells that secrete anti RBP antibodies were selected in HAT medium. One of them was identified as Ig Gl and the rest are on the study. According to the average hydrophilicity values of RBP there might be at least eight antigenic determinants and its signal sequence is foled to the inner space. This fact suggest that monoclonal antibodies against RBP can detect any conformational change between precursor and mature form of RBP.