A solid phase radiommunoassay on hydrophobic membrane filters: detection of antibodies to gonococcal surface antigens.
A solid phase radioimmunoassay (SPRIA) has been developed for detection of IgG antibodies to gonococcal outer membrane components. Gonococcal antigens were immobilized on a solid support by covalent coupling to CNBr-activated Sepharose in the presence of the detergent Triton X-100. Binding of specific antibody to the Sepharose-antigen complex was detected using radiolabelled Protein A as the antiglobulin. Protein A was labelled by radioacetylation with tritiated acetic anhydride, yielding a product of high specific activity and high stability. No detectable loss of activity was observed over a ten month period. The entire assay was performed on Mitex teflon hydrophobic membrane filters which held the Sepharose beads and aqueous supernatant as a discrete drop of liquid. The supernatants and incubation media were easily and rapidly removed from the beads by suction on a specially designed manifold system. This procedure removed the need for repeated and time-consuming centrifugations. Titres were obtained graphically from double log plots of cpm bound versus antiserum dilution by extrapolation of the straight line to a point corresponding to twice the control level of radioactivity binding. The assay proved to be a very reliable and simple procedure for the detection of IgG antibodies to gonococcal surface antigens.
D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D002462:Cell Membrane / Q000276:immunology; D005374:Filtration / Q000295:instrumentation; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008567:Membranes, Artificial; D009344:Neisseria gonorrhoeae / Q000276:immunology*; D011863:Radioimmunoassay; D012685:Sepharose; D013205:Staphylococcal Protein A / Q000494:pharmacology
D008070:Lipopolysaccharides; D008567:Membranes, Artificial; D013205:Staphylococcal Protein A; D012685:Sepharose