Monoclonal antibodies specific for oligosaccharides prepared by partial nitrous acid deamination of heparin.
Monoclonal antibodies were raised against a conjugate between heparin oligosaccharides and human serum albumin. The oligosaccharides were prepared by partial nitrous acid degradation of heparin and were coupled to human serum albumin by reductive amination. Characterization of the antibodies secreted by one of the resulting clones showed that they recognize a determinant present in the oligosaccharide antigen, but not in intact heparin, nor in a variety of related polysaccharides. Degradation of heparin by nitrous acid generates a 2,5-anhydro-D-mannose residue at the reducing end of the resulting oligosaccharides, and it is concluded that this structure is essential for interaction with the antibodies. Reduced oligosaccharides (containing a terminal anhydromannitol residue) are also active. After gel chromatography of partially degraded heparin, the smallest components capable of binding to the antibodies were found in a tetrasaccharide fraction. Affinity chromatography on immobilized monoclonal antibodies separated this tetrasaccharide fraction into distinct populations of binding and nonbinding species. Structural analysis showed that the tetrasaccharide fraction that bound to the monoclonal antibodies contained one single component with the structure IdoA(2-OSO3)-GlcNSO3 (6-OSO3)-IdoA(2-OSO3)-aManR(6-OSO3), whereas the fraction that did not bind to the antibodies contained a mixture of different structures.
D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000032:analysis*; D006493:Heparin / Q000032:analysis / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009573:Nitrites / Q000378:metabolism*; D009608:Nitrous Acid / Q000378:metabolism*; D009844:Oligosaccharides / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009573:Nitrites; D009844:Oligosaccharides; D006493:Heparin; D009608:Nitrous Acid