Generation of monoclonal antibodies against the Galβ1-4Gal epitope: a key tool in studies of species-specific glycans expressed in fish, amphibians and birds.

Whereas the Gal beta 1-4Gal epitope is rarely found in mammalian glycans, it has been found in glycans of various species of non-mammalian vertebrates, such as fish, amphibians and birds. Although glycans containing Gal beta 1-4Gal in these vertebrates were detected by precise structural analysis of the glycans using mass spectrometry and/or NMR spectrometry, there are no convenient methods to detect Gal beta 1-4Gal from various samples. To analyze systematically the distribution of Gal beta 1-4Gal in nature, we generated mouse monoclonal antibodies (mAbs) specific for Gal beta 1-4Gal using extracts of medaka eggs as an immunogen. Four mAbs (two immunoglobulin (Ig)Ms and two IgG1s) were obtained by enzyme-linked immunosorbent assay-based screening. The specificities of these mAbs were evaluated by frontal affinity chromatography using 142 kinds of 2-aminopyridine (PA)-derivatized oligosaccharides. While all mAbs interacted with (Gal beta 1-4Gal)-containing oligosaccharides at their non-reducing termini with dissociation constants (K-d) ranging from 1.0 x 10(-5) to 2.8 x 10(-4) M, no apparent interaction was observed with any other glycans. The number of branches containing Gal beta 1-4Gal on N-glycans did not significantly affect K-d of mAbs of IgG1 subclasses, but those of IgM mAbs were decreased by similar to 1 order of magnitude, in increments of the number of branches present. Using the mAbs, we established that Gal beta 1-4Gal is also expressed on glycoproteins in various tissues from the African clawed frog. Immunohistochemical staining of medaka sections revealed that Gal beta 1-4Gal epitopes were expressed in the endothelium, epithelium and epidermis, which directly contact the external environment or invading organisms. Thus, these mAbs are useful for systematically investigating the species-specific expression of glycans, which may act as a barrier against infection.