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 enzyme-linked immunosorbent assay (ELISA), is difficult because they are hydrophilic. We developed four new methods for the immobilization of oligosaccharides. ELISA plates were first coated with methyl vinyl ether-maleic anhydride copolymer (MMAC) and an excess of active anhydride groups was introduced. They were subsequently reacted, in four different ways, to bind oligosaccharides. In method 1, the anhydride groups were reacted with hydrazide groups, in the presence of adipic acid dihydrazide, and then coupled to the reducing ends of sugar chains by reductive amination. In method 2, the anhydride groups were reacted with p-aminophenyl glycoside obtained by reduction with p-nitrophenyl glycoside. In method 3, the anhydride groups were reacted with 1, 6-hexamethylenediamine. Aminooxy groups were coupled to the amino groups introduced and then aminooxyacetic acid with carbodiimide and ligated to oligosaccharides by oxime formation. In method 4, stereospecifically aminated oligosaccharides reacted with the anhydride groups. We compared, in solid-phase assays systems, the ability of lectins to detect oligosaccharides immobilized with either one of these four new methods or one of the two methods previously described. Detection of sugars with lectins is useful because, in most cases, they recognize sugars stereospecifically. The immobilization method should therefore be carefully selected to avoid changing the configuration and substitution in C-1.
Satoh
A
A
Department of Chemistry, Faculty of Science, Ochanomizu University, Otsuka 2-1-1, Bunkyo-ku, Tokyo, 112-8610, Japan.
Fukui
E
E
Yoshino
S
S
Shinoda
M
M
Kojima
K
K
Matsumoto
I
I
D002241:Carbohydrates / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*
D002241:Carbohydrates