Two mouse hybridoma antibodies against human milk-fat globules recognise the I(MA) antigenic determinant β- d-Gal p-(1→4)-β- d-Glc pNAc-(1→6)

Abstract

Two mouse hybridoma antibodies (LICR-LON-M39 and LICR-LON-M18) against the human-milk-fat globules were found to resemble human autoantibodies of anti-I type in their cold agglutinating property and their preferential reactions with erythrocytes of I- rather than i-type. From inhibition of binding assays with glycoproteins having known A, B, H, Le a, Le b, I, and i activities, and oligosaccharides of the Type 1 and Type 2 lacto-N-glycosyl series, it was established that these antibodies are directed at Type 2 structures, and that the I(Ma) determinant, β- d-Gal p-(1→4)-β- d-Glc pNAc-(1→6), which is usually found on branched oligosaccharides, is the preferred sequence. The hybridoma antibodies as well as anti-I Ma were shown to react well with the β- d-Gal p-(1→4)-β- d-Glc pNAc-(1→6)- d-Gal or - d-Man sequence. Studies of the reactions of these antibodies with glycolipids on thin-layer plates showed that the two hybridoma antibodies differ from anti-I Ma in reacting weakly with the unbranched i-type sequence β- d-Gal p-(1→4)-β- d-Glc pNAc-(1→3)-β- d-Gal p-(1→4)- β- d-Glc pNAc-(1→3)-β- d-Gal p-(1→4) as found on lacto-N-norhexaosylceramide. Furthermore, they differ from anti-I Ma but resemble anti-I Woj and Sti, and a hybridoma antibody 1B2 in their failure to react with their determinant in the presence of α- d-(1→3)-linked galactosyl groups. From their lack of reactions with blood-group-A and -H active glycoproteins, and their reactions with neuraminidase-treated erythrocytes, it was deduced that the determinants recognised by the two hybridoma antibodies are also masked in the presence of α- l-(1→2)-linked focosyl groups and sialic acid.