Production of an antibody against guinea pig MIF: II. Analysis of the antibody-reacting material using radiolabeled lymphokines

Abstract

An antibody rasied in a rabbit against guinea pig macrophage migration inhibitory factor (MIF) was proved to bind specifically to MIF, but not to three other lymphokines, macrophage chemotactic factor (MCF), neutrophil chemotactic factor (NCF), and skin reactive factor (SRF). In this study, the material which reacted with the antibody was analyzed using radiolabeled lymphokines. First, the postlabeled lymphokine or control preparation with tritiated N-succinimidyl propionate was applied onto the immunoadsorbent columns. The materials recovered from the columns were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By this method, the antibody appeared to react with a homogeneous protein in terms of its molecular weight (MW), 35,000. Second, the double-labeled lymphocyte culture supernatants were used, in which supernatants of stimulated and unstimulated cultures were labeled with either [ 3H]- or [ 14C]leucine, respectively. By this method, also a single polypeptide with a MW of 35,000 and an isoelectric point of p I 5.0–5.5, was detected. These results indicate that the protein with a MW of 35,000 and an isoelectric point of p I 5.0–5.5 may be MIF.