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Solid Phase Radioimmunoassay for Quantitation of Antigen-Specific IgG in Human Sera with ¹²⁵I-Protein A from Staphylococcus Aureus¹

From the Johns Hopkins University, School of Medicine, Department of Medicine, Division of Clinical Immunology, Baltimore, Maryland 21239

Abstract

Radiolabeled protein A from Staphylococcus aureus (Staph A) has been used to develop a solid phase, noncompetitive radioimmunoassay for quantitation of specific IgG antibody. The assay involves two incubations: First, agarose-insolubilized antigen is mixed with serum samples for 1 to 4 hr during which specific antibody is bound; second, after a washing procedure, the solid phase immune complexes are incubated for 4 to 18 hr with ¹²⁵I-Staph A, during which the radiolabeled detection protein binds to the insolubilized specific IgG antibody. In a comparative study of the IgG antiphospholipase A antibody content of 23 human sera drawn from honeybee venom-sensitive patients, results of the Staph A assay correlated highly (r = 0.981, p < 0.001, N = 23) with those obtained from a liquid phase, competitive radioimmunoprecipitation (double antibody) assay. The two assays demonstrated comparable precision, sensitivity, and reproducibility. In contrast, the use of ¹²⁵I-Staph A in the solid phase radioimmunoassay was supperior to ¹²⁵I rabbit anti-human IgG because of lower negative serum (blank) values, shorter time required to reach equilibrium binding, and greater precision and reproducibility. In principle, the ¹²⁵I Staph A assay may be applied to IgG quantitation for crude allergen extracts as well as purified antigens. Furthermore, the sera of a number of mammalian species may be studied without further modification.

Footnotes

¹ This work was supported by Grants AI 11936 and AI07290 from the National Institute of Allergy and Infectious Diseases. This is Publication 326 of the O'Neill Research Laboratories, The Good Samaritan Hospital, 5601 Loch Raven Boulevard, Baltimore, Maryland 21239.

² Correspondence to: Robert Hamilton, Johns Hopkins University School of Medicine at Good Samaritan Hospital, 3N O'Neill Laboratories, 5601 Loch Raven Boulevard, Baltimore, Maryland 21239.