Production and characterization of monoclonal antibodies specific to lactotriaosylceramide

We have established hybridoma cell lines producing monoclonal antibodies (mAbs) directed to N-acetylglucosaminyl beta 1-3galactose (GlcNAc beta 1-3Gal) residue by immunizing BALB/c mice with lactotriaosylceramide (Lc(3)Cer). These obtained hybridoma cells, specific to Lc(3)Cer, were dual immunoglobulin (Ig)-producing cells which secreted both IgM and IgG molecules as antibodies. The established mAbs are able to react with not only Lc(3)Cer but also GlcNAc beta 1-3-terminal glycosphingolipids (GSLs) despite branching or lactosamine chain lengths and human transferrin with terminal GlcNAc residues. Comparison of the variable regions of the cloned IgM and IgG by reversed transcription-polymerase chain reaction analysis confirmed that the variable regions determine the specificity, the other amino acids are conserved, and these mAbs are encoded by J558 and V kappa-21family genes. Furthermore, we have analyzed the expression of GSLs with GlcNAc beta 1-3 epitope in acute leukemia cell lines and mouse fetal tissues using these mAbs, in which antigens were distributed comparatively. These mAbs are useful for studying the precise distribution of GlcNAc beta 1-3Gal-terminating GSL expression in tissues as well as for detecting GSLs carrying terminal GlcNAc beta 1-3Gal carbohydrate structure.