Usefulness of synthetic Mimetic ligands in establishing an affinity chromatography procedure for purifying estradiol antibody in sheep serum: potential application in regulating fertility of the ram.

Recently developed synthetic Mimetic affinity ligands were screened for their ability to purify estradiol antibody from sheep serum. One of these ligands (color coded Yellow 1) effectively separated immunoglobulin from other serum protein by negative affinity. When 1 ml antiserum diluted 70% (protein content approximately 25 mg) was passed through 1 ml gel with Yellow 1 ligand, estradiol antibody-binding activity (% binding of E2 tracer) peaked in the effluent (62%) and decreased progressively in the subsequent three 4-ml fractions of the wash (34%, 6% and 2%, respectively). As long as the total amount of serum protein applied remained below the saturable-binding capacity of the ligand (determined to be approximately 28 mg), the relative purity of estradiol antibody in the effluent (E2 binding per unit protein) remained very high with only 3% (0.78 mg) of applied protein appearing in the effluent. Purity of antibody in the effluent was confirmed by electrophoresis carried out on SDS polyacrylamide gel. Flow rate of serum diluted 42% (1 ml, protein content approximately 48 mg) was constant and acceptable (0.37 ml/min) and uncoupling of nonimmunoglobulin serum protein from the ligand was complete by the time the column was reequilibrated. We conclude that of the different Mimetic ligands evaluated Yellow 1 has properties that would be useful in a large-scale affinity chromatography procedure for purifying immunoglobulin from sheep serum.