Selective biotinylation of Neisseria meningitidis group B capsular polysaccharide and application in an improved ELISA for the detection of specific antibodies.
A method is described for the selective biotinylation of meningococcal capsular polysaccharide from Neisseria meningitidis group B and its application to an enzyme-linked immunoabsorbent assay (ELISA) to detect specific antibodies by immobilization on streptavidin-coated microtiter wells. Capsular polysaccharide from Neisseria meningitidis B has been biotinylated by specific periodate oxidation of terminal residues and condensation of the resulting aldehydes with biotin hydrazide, using a spin-column technique in the intermediate purification steps. The ELISA was optimized employing an extended reaction time between the label alkaline phosphatase and its most common substrate, p-nitrophenyl phosphate, together with evaluation of blocking agents to minimize non-specific binding. Specificity was demonstrated by a direct competitive enzyme immunoassay (EIA).
Diaz Romero
J
J
Servicio de Inmunologia, Centro Nacional de Microbiologia (C.N.M.V.I.S), Majadahonda, Madrid, Spain.
Outschoorn
I
I
D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis*; D016667:Bacterial Capsules / Q000276:immunology*; D001710:Biotin; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D009345:Neisseria meningitidis / Q000276:immunology*; D011135:Polysaccharides, Bacterial / Q000276:immunology*; D012680:Sensitivity and Specificity
D000907:Antibodies, Bacterial; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011135:Polysaccharides, Bacterial; D001710:Biotin