G. Brian Wisdom

Conjugation of Antibodies to Horseradish Peroxidase

Horseradish peroxidase is coupled to IgG antibody in a two-step procedure. In the first step monosaccharide residues in the enzyme are oxidized with periodate to produce aldehyde groups. Then, in the second step, the aldehyde groups are allowed to react with amino groups in the IgG antibody. The Schiff bases formed are reduced and the conjugate is purified by gel filtration.

Conjugation of Antibodies to Fluorescein or Rhodamine

The isothiocyanate derivative of the fluorophore, fluorescein or rhodamine, is coupled to the amino groups of IgG antibody in a one-step procedure and excess label is removed by gel filtration.

The determination of asialoglycoforms of serum glycoproteins by lectin blotting with Ricinus communis agglutinin

Serum proteins were fractionated by polyacrylamide gel electrophoresis under denaturing conditions and transferred to nitrocellulose membranes. The blotted polypeptides were probed with biotinylated Ricinus communis lectin (RCA120) followed by streptavidin/alkaline phosphatase. This procedure detected five asialoglycoproteins (alpha 2-macroglobulin, transferrin, alpha 1-antitrypsin, alpha 1-antichymotrypsin and haptoglobin beta chain). The asialoform of the alpha 1-trypsin inhibitor was found to be decreased in inflammation.

Conjugation of Antibodies to Alkaline Phosphatase

Alkaline phosphatase is coupled to immunoglobin G antibody in a one-step procedure using the homobifunctional reagent glutaraldehyde, which reacts with amino groups in the two proteins. The procedure is simple to perform and requires minimal equipment.