caa a22 4500 465734413 CHVBK 20180323111750.0 cr unu---uuuuu 170327e19900501xx s 000 0 eng 10.1007/BF01050604 doi (NATIONALLICENCE)springer-10.1007/BF01050604 Preparation of polysaccharide-enzyme conjugates for competitive binding assays [Elektronische Daten] [Peter Meikle, David Bundle] A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to β-galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme. Glycoconjugate Journal AB, 1990 enzyme glycoconjugate nationallicence enzyme labelled O-polysaccharide nationallicence enzyme immunoassay nationallicence reductive amination nationallicence Meikle Peter Division of Biological Sciences, National Research Council of Canada, K1A OR6, Ottawa, Ontario, Canada aut Bundle David Division of Biological Sciences, National Research Council of Canada, K1A OR6, Ottawa, Ontario, Canada aut Glycoconjugate Journal Kluwer Academic Publishers 7/3(1990-05-01), 207-218 0282-0080 7:3<207 1990 7 10719 https://doi.org/10.1007/BF01050604 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF01050604 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Meikle Peter Division of Biological Sciences, National Research Council of Canada, K1A OR6, Ottawa, Ontario, Canada aut NATIONALLICENCE 700 1- Bundle David Division of Biological Sciences, National Research Council of Canada, K1A OR6, Ottawa, Ontario, Canada aut NATIONALLICENCE 773 0- Glycoconjugate Journal Kluwer Academic Publishers 7/3(1990-05-01), 207-218 0282-0080 7:3<207 1990 7 10719 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer