Immunochemical characterization with monoclonal antibodies of three major caseins and alpha-lactalbumin from rat milk

Abstract

Rat milk contains at least three major caseins with apparent molecular weights of 41,000 (alpha-casein), 25,000 (beta-casein), and 22,000 (gamma-casein) (estimated in 10% sodium dodecyl sulfate-polyacrylamide gels). These three caseins and alpha-lactalbumin, a major whey protein, were purified from rat milk. The purified caseins and alpha-lactalbumin were used to immunize BALB/c mice, and spleen cells from these mice were hybridized with cells of the mouse myeloma SP-2/0 cell-line. We have isolated a small library of hybridoma cell-lines secreting monoclonal antibodies specific for each of the major caseins and alpha-lactalbumin from rat milk. Antibodies were tested for immunoreactivity with each of the purified milk proteins and with total rat milk proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Some heterogeneity in apparent molecular weight was observed for purified alpha-casein, gamma-casein, and alpha-lactalbumin. Monoclonal antibodies against alpha-casein, gamma-casein, and alpha-lactalbumin recognized all of the molecular weight forms of the antigen for which they were specific. Each monoclonal antibody was specific for one of the caseins or alpha-lactalbumin and did not react with the other caseins or alpha-lactalbumin, suggesting that there is limited structural homology among these proteins. All of the monoclonal antibodies against the rat caseins reacted with components of mouse milk, and the monoclonal antibodies against rat gamma-casein reacted with a component of human milk of apparent molecular weight 27,000. No interspecies reactivity was observed with the antibodies against rat alpha-lactalbumin. These monoclonal antibodies are being used to develop sensitive assays for each of these major rat milk proteins.