NEW METHOD USING P-BENZOQUINONE FOR COUPLING ANTIGENS AND ANTIBODIES TO MARKER SUBSTANCES

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  • 1 January 1976
    • journal article
    • research article
    • Vol. C127  (2) , 197-208
Abstract
A method using p-benzoquinone for coupling antigens and antibodies to enzymes and erythrocytes is described. The method involves the treatment of proteins (or polysaccharides) at pH 6 or 7 with an excess of p-benzoquinone. After removal of the unreacted reagent by gel filtration, the activated proteins were coupled at pH 8-9 with enzymes or erythrocytes. Biological activities of the proteins were not substantially modified by this treatment since 80-100% of the antigen binding capacity was preserved in p-benzoquinone treated antibodies or Fab fragments. Anti-Ig [immunoglobulin] antibodies (or Fab) were coupled by this procedure to peroxidase, alkaline phosphatase, lactoperoxidase, glucose oxidase and .beta.-galactosidase, and the conjugates obtained were highly effective in detecting intracellular Ig by immunohistochemical techniques. Erythrocytes coated with sheep anti-mouse Ig antibody or Fab were used to titrate by passive hemagglutination serum Ig. The same erythrocytes were employed to detect by plaque assay mouse Ig secreting cells. Erythrocytes coated with peroxidase, alkaline phosphatase, bovine serum albumin, RNase, Salmonella polysaccharide (B 27 +) and pneumoccocal polysaccharide SIII were employed to titrate serum antibody by passive hemagglutination and hemolysis and to detect mouse antibody secreting cells by plaque assay. All the antigen and antibody coated erythrocytes prepared gave highly satisfactory and reproducible results.