Anti-nitroxide immunoglobulin G: analysis of antibody specificity and their application as probes for spin-labeled proteins.
Antibodies have been elicited to the nitroxide spin-label 4-maleimido-2,2,6,6-tetramethyl-piperidinyl-1-oxy conjugated, via protein sulfhydryl groups, to bovine serum albumin. Antibody-hapten cross-reactivity was demonstrated by double immunodiffusion and by a broadening of the nitroxide electron paramagnetic resonance spectrum. The specificity of the antibodies with respect to hapten structure was examined by means of a simple filter binding assay. Under these conditions, antibodies were shown to distinguish between the nitroxide and hydroxylamine derivatives and between spin-labels comprising either five- or six-membered ring structures. In addition, protein-bound nitroxide spin-labels were detected at the nanogram level by immunoblotting. By use of this method, the specificity of the antibody-hapten reaction predicted by the filter binding assay procedure was utilized to differentially detect various types of bound spin-label. Finally, antibodies were used to identify protein-bound nitroxide spin-label of protein fractionated by gel electrophoresis.
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002417:Cattle; D003429:Cross Reactions; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D006241:Haptens; D005779:Immunodiffusion; D007074:Immunoglobulin G; D011817:Rabbits; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; D013113:Spin Labels
D000906:Antibodies; D000936:Antigen-Antibody Complex; D003497:Cyclic N-Oxides; D006241:Haptens; D007074:Immunoglobulin G; D012709:Serum Albumin; D013113:Spin Labels; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl; D012710:Serum Albumin, Bovine