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The Journal of Immunology, 1971, 106: 1630-1640.
Copyright © 1971 by The American Association of Immunologists, Inc.
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The Detection of Antibody to Hemocyanin by Direct Solid-Phase (Polystyrene) Radioimmunoassay and Gel Filtration1

Angeliki O. Kotoulas and Leonard A. Moroz2

From the Harry Webster Thorp Laboratories, Division of Immunochemistry and Allergy, McGill University Clinic, Royal Victoria Hospital, Montreal, Quebec, Canada

Abstract

A sensitive, relatively simple method is described for the direct solid-phase radioimmunoassay of antibody to a macroglobulin antigen, keyhole limpet hemocyanin (KLH). Antibody is adsorbed to polystyrene tubes (antibody-binding phase), reacted with 125I-KLH (antigen-binding phase) and excess 125I-KLH removed by washing, the residual radioactivity providing a measure of antibody activity. The sensitivity of the method (5 to 10 ng antibody protein) is comparable to other primary antibody assays, and is markedly dependent on ionic strength and total protein concentration during the antibody-binding phase. The assay is equal to or slightly less sensitive than passive hemagglutination for detecting 19S antibody, and consistently more sensitive for detecting 7S antibody. A method of similar sensitivity is also described for the detection of anti-KLH antibody by gel filtration on agarose columns. Both methods are potentially applicable to the detection of antibody to other, particularly macroglobulin, antigens.

Footnotes

1 This work was supported by grants from the Medical Research Council of Canada, the Quebec Medical Research Council and the Muscular Dystrophy Association of Canada.

2 Medical Research Scholar, Medical Research Council of Canada.






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