Isolation of specific oligoclonal antibodies against bovine αs1-casein by FPLC tandem immunoaffinity of the polyclonal antibodies

Abstract

Polyclonal antibodies specifically directed towards native casein fractions have been recently used to solve some analytical problems such as localization of casein antigenic sites (Otani et al. 1985; Ametani et al. 1987), identification of casein variants (Moio et al. 1989a; Chianese et al. 1992), detection of bovine casein adulterating goats', ewes' and water-buffalo milk and cheese (Elbertzhagen & Wenzel, 1982; Bernhauer et al. 1983; Aranda et al. 1988; Moio et al. 1992; Rolland et al. 1993, 1995; Addeo et al. 1995b), evaluation of the efficiency of chromatographic fractionation of casein (Addeo et al. 1992) and detection of casein proteolysis in cheese (Addeo et al. 1995a). However, the use of polyclonal antibodies for immune analysis is often limited owing to nonspecific binding. The use of monoclonal antibodies may overcome this problem to some extent. Although the binding affinity of a hyperimmune serum is seldom attainable with a monoclonal antibody, an alternative approach consists of the isolation of specific antibodies by affinity chromatography (Johnstone & Thorpe, 1982). In a single step 1000–10000-fold purification has been achieved.In this paper a fast protein liquid chromatography (FPLC) tandem immunoaffinity is used to enhance the specificity of bovine αs1-casein (CN) polyclonal antiserum. In the first column a caprine whole casein lacking αs1-CN was bound to a solid phase matrix and in the second column bovine αs1-CN was bound. After elution of macromolecular contaminants by a washing step, the two columns were detached and the purified bovine αs1-CN antibodies were eluted from the second column by a simple pH change.