The use of protein A in solid-phase binding assays: a comparison of four radioiodination techniques.
Preparations of protein A radioiodinated by 4 different methods have been compared in indirect radioimmunoassays. The oxidative methods (chloramine-T and iodogen) for direct iodination of tyrosyl and histidyl residues were applied with high efficiency and gave a suitable product, provided the substitution ratio was kept low (1 iodine atom/molecule of protein A). Higher levels of modification tended to perturb the Fc-binding characteristics of the protein, especially with the use of iodogen. Introduction of the isotope via substitution of lysyl residues (Bolton-Hunter and Wood reagents) was also examined. The Bolton-Hunter modification of protein A gave an unsuitably low labeling efficiency; in contrast, the Wood reagent gave efficiencies approaching 50%. Protein A could be extensively substituted with the latter reagent (greater than 5 diiodinated benzimidate molecules per protein molecule). Thus, the use of the Wood-labeled protein A could raise the sensitivity of the binding assay at least an order of magnitude compared to using protein A iodinated by the oxidative methods. The effects on the biological activity of protein A exerted by the different labeling procedures are rationalized on the basis of the amino acid composition and tertiary structure of the protein.
D002700:Chloramines; D006639:Histidine; D007467:Iodoproteins / Q000276:immunology*; D008239:Lysine; D013205:Staphylococcal Protein A / Q000276:immunology; D013329:Structure-Activity Relationship; D014443:Tyrosine
D002700:Chloramines; D007467:Iodoproteins; D013205:Staphylococcal Protein A; D014443:Tyrosine; D006639:Histidine; D008239:Lysine