A novel preparation of immunoadsorbents. Controlled coupling of proteins to Sepharose-gelatin by heterobifunctional reagent.
The heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was utilized for controlled coupling of lysozyme and bovine serum albumin to Sepharose-gelatin. Initially the protein (lysozyme or bovine serum albumin) was reacted with N-succinimidyl 3-(2-pyridyldithio)propionate at the free amino groups to give 3-(2-pyridyldithio)propionyl-protein. The latter was reduced to thiopropionyl-protein and was conjugated to 3-(2-pyridyldithio)-propionyl-Sepharose-gelatin through sulfhydryl-disulfide exchange. Sepharose-gelatin-lysozyme and Sepharose-gelatin-albumin were prepared in this manner. They were capable of binding their respective antibody and the eluted antibody was found to be pure on electrophoresis in SDS-polyacrylamide gels and to show heterogeneity by isoelectric focusing. Antibodies bound to Sepharose-gelatin-albumin were found to be less tightly bound to the immunoadsorbent than in the case of Sepharose-albumin, as more antibodies were eluted on the former immunoadsorbent with 0.1 M glycine-HCl (pH 3) than on the latter. The new method permits controlled coupling of proteins to an insoluble matrix (Sepharose-gelatin), and the bond through which reaction occurs is known with precision.
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002417:Cattle; D005780:Gelatin; D007164:Immunosorbents; D007202:Indicators and Reagents; D009113:Muramidase; D010084:Oxidation-Reduction; D011485:Protein Binding; D011506:Proteins; D011725:Pyridines; D012685:Sepharose; D012710:Serum Albumin, Bovine; D013388:Succinimides
D000906:Antibodies; D000936:Antigen-Antibody Complex; D007164:Immunosorbents; D007202:Indicators and Reagents; D011506:Proteins; D011725:Pyridines; D013388:Succinimides; D012710:Serum Albumin, Bovine; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D005780:Gelatin; D012685:Sepharose; D009113:Muramidase