Four rabbit antibodies have been prepared, which are specifically directed against a-lactalbumins from different sources; namely human, cow, goat and sheep milk. Each of these antibodies was tested for its ability to react with, separately, each of the four proteins.The immunological reactions were assessed by means of different techniques : double immunodiffusion in agar gel as well as affinity chromatography of antibodies, using antigens covalently bound to an insoluble matrix. In each case, the strongest reaction was observed between homologous antibody and (matrix-bound) antigen ; heterologous antigens were, however, also capable of cross-reaction.Whereas no cross-reaction between human cc-lactalbumin and antibodies against the bovine protein could be evidenced by immunodiffusion, the occurrence of soluble complexes has been disclosed by means of a gel filtration technique.It has been reported that antisera to bovine a-lactalbumin react with a-lactalbumins from other ruminants, but fail to do so with the parent proteins from non-ruminants [l, 21. However, when the amino acid sequences of human, of bovine and of guinea-pig a-lactalbumins became available [3 -51, their comparison afforded evidence for a strikingly high degree of homology at the structural level. This was difficult to reconcile with the view that the antigenic sites of bovine and human a-lactalbumins, or guineapig, should all be different.Many studies indeed have already demonstrated immunological cross-reactions of iso-functional enzymes from different species. Such is for example the case with lysozymes from duck and from hen egg white [6], or with bovine and porcine trypsins [7]. In this respect, the pattern of cross-reactions between different a-lactalbumins would appear to be anomalous or, possibly, it might result from the experimental procedure that was used.In earlier work, the immunological comparison of a-lactalbumins was in fact performed by means of quantitative, single immunodiffusion, a method that precludes the disclosure of eventual soluble antigen-antibody complexes.Abbreviation. Mops, morpholino-propane sulfonic acid.
Eur. J. Biochem. 50 (1975)The present investigation was therefore undertaken in order to find out if antigenic sites of human, of bovine, of goat and of sheep a-lactalbumins are not able to cross-react, to some detectable extent, with heterologous antibodies.
EXPERIMENTAL PROCEDURE
MaterialsSepharose 4B, Sephadex G75 and DEAE-Sephadex A25 were purchased frorn Pharmacia Fine Chemicals (Uppsala, Sweden). Cyanogen bromide was from Fluka. Freund's complete adjuvant was a Difco product. All other reagents were of analytical grade.Gradients for chromatography were set up with an LKB Ultrograd 11 300 unit, monitored by an LKB Uvicord 11.
Purification of a-LactalbuminsThe purification of human a-lactalbumin was carried out according to the procedure already described by Findlay and Brew [3].Bovine, goat and sheep a-lactalbumins were purified by the procedure of Schmidt and Ebner [8], up to the ammonium sulphate precipita...
