Comparison of methods of immobilization to enzyme-linked immunosorbent assay plates for the detection of sugar chains

The immobilization of carbohydrates for solid-phase assays, including enzym e-linked immunosorbent assay (ELISA), is difficult because they are hydroph ilic. We developed four new methods for the immobilization of oligosacchari des. ELISA plates were first coated with methyl vinyl ether-maleic anhydrid e copolymer (MMAC) and an excess of active anhydride groups was introduced. They were subsequently reacted, in four different ways, to bind oligosacch arides. In method 1, the anhydride groups were reacted with hydrazide group s, in the presence of adipic acid dihydrazide, and then coupled to the redu cing ends of sugar chains by reductive amination, In method 2, the anhydrid e groups were reacted with p-aminophenyl glycoside obtained by reduction wi th p-nitrophenyl glycoside. In method 3, the anhydride groups were reacted with 1,6-hexamethylenediamine. Aminooxy groups were coupled to the amino gr oups introduced and then aminooxyacetic acid with carbodiimide and ligated to oligosaccharides by oxime formation. In method 4, stereospecifically ami nated oligosaccharides reacted with the anhydride groups. We compared, in s olid-phase assays systems, the ability of lectins to detect oligosaccharide s immobilized with either one of these four new methods or one of the two m ethods previously described. Detection of sugars with lectins is useful bec ause, in most cases, they recognize sugars stereospecifically. The immobili zation method should therefore be carefully selected to avoid changing the configuration and substitution in C-1. (C) 1999 Academic Press.