Immunochemical evaluation of radioiodinated protein A.
The [125I]iodoprotein A iodinated by the chloramine-T method using carrier-free 125I- to various specific activities (0.02-2.0 125I/mole protein A) displayed 85% binding activity to excess solid-phase rabbit IgG. On storage, however, the solid-phase IgG binding activity of the high specific activity [125I]iodoprotein A (approximately 2 125I/protein A) decreased rapidly while that of the low specific activity [125I]iodoprotein A (approximately 0.2 125I/protein A) remained essentially unchanged. Analysis of aged high specific activity [125I]iodoprotein A by thin-layer chromatography revealed that free 125I- was released from the [125I]iodoprotein A during storage. Removal of the released 125I- was accomplished by using anion exchange resin. High specific activity [125I]iodoprotein A was found to be more sensitive than the low specific activity [125I]iodoprotein A in quantitating aggregated IgG while the latter was as useful as the former in hybridoma screening. When protein A was labeled with the Bolton-Hunter reagent, the [125I]iodoprotein A had an IgG binding property and stability similar to the [125I]iodoprotein A labeled with the chloramine-T method. Since the chloramine-T method can yield high specific activity [125I]iodoprotein A and repurification of the aged preparation can easily be achieved by using anion exchange resin, we concluded that chloramine-T is more efficient than Bolton-Hunter reagent and that both methods yield materials with comparable immunochemical properties.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis; D002700:Chloramines; D004355:Drug Stability; D007074:Immunoglobulin G / Q000378:metabolism; D007457:Iodine Radioisotopes; D051379:Mice; D010406:Penicillins / Q000276:immunology; D011485:Protein Binding; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods*; D013205:Staphylococcal Protein A / Q000378:metabolism*; D014105:Tosyl Compounds
D000911:Antibodies, Monoclonal; D002700:Chloramines; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D010406:Penicillins; D013205:Staphylococcal Protein A; D014105:Tosyl Compounds; C016300:chloramine-T