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Enzyme-Linked Immunosorbent Assay, Elisa

III. Quantitation of Specific Antibodies by Enzyme-Labeled Anti-Immunoglobulin in Antigen-Coated Tubes¹

From the Wenner-Gren Institute, Department of Immunology, University of Stockholm, Norrtullsgatan 16, S-113 45 Stockholm, Sweden

Abstract

A sensitive and simple method for the quantitative determination of antibodies is reported. Tubes coated with antigen are incubated with antiserum followed by an enzyme-labeled preparation of anti-immunoglobulin. The enzyme remaining in the tubes after washing provides a measure of the amount of specific antibodies in the serum. Coating of polystyrene tubes with antigen is described, as well as the preparation of specifically purified antibodies against rabbit IgG, and their conjugation to alkaline phosphatase.

When rabbit antisera against human serum albumin or against the dinitrophenyl group (DNP) were incubated in tubes coated with antigen, less than 1 ng/ml of specific antibody could be detected in both systems. Antibodies in unknown sera could be quantitated by comparison with a standard antiserum. The results obtained compared well with those obtained with other methods for antibody determination.

In the DNP system, the specificity of the reaction was assessed by inhibition with hapten. The reaction of immune serum against DNP with DNP-protein, adsorbed to the tubes, was completely inhibited by hapten in solution.

Footnotes

¹ This work was supported by Grant 2032-035 from the Swedish Natural Science Research Council and by a grant from Pharmacia AB, Uppsala, Sweden.