Enzyme Immunoassay for Antibodies in Serum Using a Covalent Chromatographic Method for Separation of the Bound Label

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Abstract
A sensitive enzyme immunoassay system for the measurement of antibodies in serum was developed by the use of a separation method based on the thiol-disulfide interchange reaction. Serum samples including antibodies (anti-insulin, anti-human chorionic gonadotropin, or anti-B-D-galactosidase) were incubated with antigens labeled with β-D-galactosidase from Escherichia coli. Each reaction mixture was then passed through a small column (0.1 ml) of (anti-IgG)IgG-Sepharose 4B, in which the (anti-IgG)IgG had been coupled by means of a disulfide bond. The column was washed to remove the unbound label, then the antibody-bound label was eluted from the column with a buffer containing 25 mM dithiothreitol, which cleaved the disulfide bonds between the Sepharose matrix and the anti-IgG antibody molecules. By measuring the enzyme activity in the eluate, the amount of antibodies could be determined even in a serum sample which contained antibodies corresponding to as little as 0.01 % of the standard antisera. Preliminary experiments showed that the present method can be used to detect the anti-insulin antibody in the sera from insulin-treated diabetic patients.