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Immunochemical Characterization with Monoclonal Antibodies of Three Major Caseins and Alpha-Lactalbumin from Rat Milk¹

Departments of Medicine and Biochemistry Case Western Reserve University and Veterans Administration Medical Center, Cleveland, OH 44106

ABSTRACT

Rat milk contains at least three major caseins with apparent molecular weights of 41,000 (-casein), 25,000 (ß-casein), and 22,000 (-casein) (estimated in 10% sodium dodecyl sulfate-polyacrylamide gels). These three caseins and -lactalbumin, a major whey protein, were purified from rat milk. The purified caseins and -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 -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 -casein, -casein, and -lactalbumin. Monoclonal antibodies against -casein, -casein, and -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 -lactalbumin and did not react with the other caseins or -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 -casein reacted with a component of human milk of apparent molecular weight 27,000. No interspecies reactivity was observed with the antibodies against rat -lactalbumin. These monoclonal antibodies are being used to develop sensitive assays for each of these major rat milk proteins.

¹ This work was supported by Grant HD-05885 from the National Institutes of Health to C.S.K. and the Research Service of the Veterans Administration.

² Department of Pathology, Case Western Reserve University.

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