Affinity Chromatography for Purification of Antibodies to Neisseria Gonorrhoeae and Neisseria Meningitidis Lipopolysaccharides

Abstract

Lipopolysaccharides (LPSs) were prepared by phenol‐water extraction of the gonococcal strain 8551 and the group B meningococcal strain 44/76, digested with pronase, and purified by ultracentrifugation and Sepharose CL‐6B fractionation in the presence of 1.5 per cent sodium deoxycholate. On SDS‐PAGE with 10 per cent acrylamide the purified 5I‐labelled LPSs migrated as single, low‐molecular‐weight components. The LPSs were coupled to CNBr‐activated Sepharose 4B for affinity purification of antibodies to the common antigenic factor 1 and the sero‐type factor 5 of LPS 8551, and antibodies to LPS 44/76. The antibodies eluted showed ELISA activity against wells coated with LPS or whole cells of the bacteria, the antibody activity being inhibited by LPS. SDS‐PAGE of whole cells of the strain 8551 and immunoblotting with the anti‐factor 1 or ‐factor 5 antibodies resulted in single, broad bands corresponding to the low‐molecular‐weight LPS subunits.

EXEMPLIFICATION OF SEROLOGICAL CROSS‐REACTIVITY OF NEISSERIA LIPOPOLYSACCHARIDES

“…(LPS) . LPS was prepared from the gonococcal strain VI1 (Gc,) by phenol-water extraction and purified by methods as reported (19). When up to 100 pg of the purified LPS was separated by sodium dodecyl sulfatepolyacrylamide electrophoresis (SDS-PAGE) and the gels were stained with Coomassie Brilliant Blue R-250, no protein line was observed.…”

“…Antisera. Antisera against whole cells of gonococci or meningococci were raised in rabbits as reported (19). IgG was isolated by a DEAE cellulose (Schleicher & Schull GmbH.…”

“…Enzymelinked immicnosorben.nr assay (ELISA) and ELISA inhibition. The reagents, microtitre plates, and other materials used were as previously reported (19). Coats were prepared with LPS (7.5 pg/ml) or with a suspension of bacteria containing approximately 2 x 10' organisms/ml.…”

“…LPS or whole-cell lysates of bacteria were separated by slab gel electrophoresis ( I5 per cent acrylamide), transferred to nitrocellulose membranes and immunoblotted against antibody. using methods described ( 19). Peroxydase-conjugated swine antibodies to rabbit IgG (DAKOPATTS.…”

Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography

“…Many approaches have been used to bind LPS or detoxified OAg from various bacteria to resins for use in affinity purification and, despite the high toxicity, CNBr-activated resin has been the most commonly employed (Stiller and Nielsen, 1983;Rodahl and Maeland, 1984). Girard and Goichot attached LPS to an aminohexyl-Sepharose resin by activation with benzoquinone (Girard and Goichot, 1981), while Fox and Hechemy tested epoxy-activated resin for the attachment of Escherichia coli LPS (Fox and Hechemy, 1978).…”