Production and characterization of antibodies to the beta-(1-->6)-galactotetraosyl group and their interaction with arabinogalactan-proteins.

Rabbit antisera were raised against beta-(1-->6)-galactotetraose coupled to bovine serum albumin (Gal4-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of alpha-L-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, beta-(1-->4)-galactan, and beta-(1-->3)-galactan, indicating their high specificity toward the consecutive beta-(1-->6)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immunobilized beta-(1-->6)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (1-->6)-linked beta-galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal4-BSA as antigen, which revealed that beta-(1-->6)-galactotriose and -tetraose were potent inhibitors, while beta-(1-->3)- or beta-(1-->4)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or beta-(1-->6)-galactotetraose.