Ann Sutton

An avidin-biotin based ELISA for quantitation of antibody to bacterial polysaccharides

A solid phase immunoassay utilizing avidin-biotin binding has been developed for measuring anticapsular polysaccharide antibodies. Capsular polysaccharides of Escherichia coli K1, Haemophilus influenzae type b, Staphylococcus aureus types 5 and 8, and levan from Aerobacter levanicum have been biotinylated through -OH or COOH groups with retention of antigenicity. Polysaccharides were immobilized on avidin-coated microtiter wells for use in an enzyme-linked immunosorbent assay (ELISA) to detect antibody. Two preparations of biotinylated S. aureus type 8 polysaccharide were equivalent as antigens in ELISA. Specificity was demonstrated by absorption of antisera, by competitive inhibition with purified antigens, and by reaction with specific monoclonal or myeloma antibodies. Reproducibility of the assay for H. influenzae type b and S. aureus type 8 antibody was demonstrated by replicate titrations of high and low level antisera.

Enzyme-linked immunosorbent assay

Publisher Summary Avidin–biotin complex formation has been used to enhance enzyme-linked immunoassay (ELISA) sensitivity by the preparation of biotinylated antibodies or indicator enzyme conjugates. The polysaccharide is derivatized with biotin via an amino or hydrazide functional group. Polysaccharides are derivatized with biotin via adipic acid hydrazide groups. The method of attachment of the hydrazide or amino groups is determined by the structure of the polysaccharide. Polysaccharide carboxyl groups are derivatized with adipic acid dihydrazide by the carbodiimide method. The degree of substitution of polysaccharides by hydrazide using either method of activation is determined by the trinitrobenzenesulfonic acid reaction. The color is developed by addition of 100 μl p -nitrophenyl phosphate to the washed plate and incubation for 30–60 minutes. Inherent with any assay method involving chemical modification of polysaccharide antigens is the risk that antibody binding can be altered. Nonspecific binding of human sera to avidin-coated plates became evident in low-titered sera. This background reactivity can be controlled by addition of sufficient antigen to coat most of the avidin sites.

771 COMPARATIVE IMMUNOGENICITY OF GROUP C NEISSERIA MENINGITIDIS VARIANTS AND ESCHERICHIA COLI K92 CAPSULAR POLYSACCHARIDES IN ADULT VOLUNTEERS

We studied three structurally and antigenically similar capsular polysaccharides: Group C Neisseria meningitidis O-acetyl positive (OAc+) and negative (OAc−) variants, and the cross-reacting E. coli K92 for their ability to induce Group C meningococcal antibodies in adults. All three polysaccharides elicited specific serum antibodies. The OAc− variant was the most immunogenic. Geometric mean pre-immunization anticapsular antibody levels were 1.4 μgm/ml, 0.8 μgm/ml, and 1.2 μgm/ml for groups receiving OAc−, OAc+ and E. coli K92 respectively. Geometric mean antibody titers 3 weeks and 2 months post immunization were 41.7 μgm/ml for OAc−, 22.8 μgm/ml for OAc+, and 7.0 μgm/ml for E. coli K92 (p = 0.001 for OAc− and OAc+ versus K92). No Group C meningococci or cross-reacting organisms were isolated from repeated NP cultures, but one individual demonstrated persistant rectal carriage of E. coli K92. Antibodies elicited by either Group C polysaccharide were bactericidal for OAc+ and OAc− organisms. Absorption of OAc+ antisera with OAc− polysaccharide did not remove all bactericidal antibody. The superior immunogenicity and distinct biochemical characteristics of the OAc− variant support further study in children and infants.