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Purification of Two Protein Antigens by Dissociation from Thiolated Antibody

From the Department of Pathobiology, Laboratory of Microbiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Abstract

A method has been devised for the purification of two protein antigens by dissociation from immune complexes containing thiolated antibody (AbSH). Precipitating antibody to bovine serum albumin (BSA) and bovine lactoglobulin (BLG) was thiolated using N-acetyl homocysteine thiolactone (AHT) at pH 10.7. After dissociation from antigen, AbSH was cross-linked and precipitated using the bifunctional organic mercurial, 3,6-bis (acetoxymercurimethyl)-dioxane (MMD). Fully active precipitating AbSH to BSA was obtained with thiolation times of 15, 30 and 60 min but not 120 min; fully active AbSH to BLG was found after 15 and 30 min but not 60 min. When the 15-min-thiolated antibody was used to purify antigen, considerable residual antibody remained associated with antigen, but this difficulty was overcome by empolying 30- and 60-min AbSH preparations. Dissociation of BSA-AbSH or BLG-AbSH complexes was best achieved at pH 1.8, using a phosphate-HCl buffer plus 40% p-dioxane. Employing the same buffer at a higher pH did not separate the AbSH completely. A glycine-H₂SO₄ buffer plus p-dioxane instead of phosphate-HCl plus p-dioxane did not dissociate antigen from AbSH. It was shown that dissociated antigen was devoid of detectable antibody, was immunoelectrophoretically homogeneous, and was recoverable specifically from mixtures of antigens in high quantity.

Footnotes

Supported by National Science Foundation Grant GB 4172 and a Career Development Award, 6K3AI6164-06, from the National Institutes of Health to Benjamin Wolf.