Production of polyclonal antibody specific for mannose-containing triglycosylceramide (Amino-CTH) and immunochemical detection of its hapten in crustacean

An antibody against Amino-CTH containing mannose, GlcNAcβ1-3 Manβ1-4 Glcβ1-Ceramide (ArOse3 Cer) isolated from the larvae of the green-bottle fly, Lucilia caesar, was purified by affinity chromatography from rabbit antiserum. The specificity of the antibody was verified by ELISA and TLC-immunostaining. It was found to bind to ArOse3Cer and ArOse3-sphingosine, and to a lesser extent to certain other compounds containing an N -acetylglucosamine residue at their termini, such as GlcNAcβ 1-3 Gal β1-4 Glcβ 1-Ceramide (Amino-CTH) and GlcNAcβ 1-2 Man α 1-3 Manβ1-4 Glcβ 1-Ceramide (MlOse4 Cer). This antibody was used to detect the natural hapten in crustacean glycolipids. The purified antibody reacted with a neutral glycosphingolipid in Euphausia superba (antarctic krill) and Macrobrachiurn nipponense (fresh-water shrimp), as indicated by TLC-immunostaining. The crustacean glycolipid antigen was isolated and characterized as GlcNAcβ1-3 Manβ 1-4 Glc-Ceramide. This is the first paper to demonstrate the presence of an Amino-CTH containing mannose in the crustacean. Cryostat sections of M. nipponense were examined to determine the distribution of ArOse3Cer with the purified antibody. Clear staining was observed in the green gland, esophagus and gills.