Monoclonal and polyclonal antibodies against porcine mitochondrial aspartate aminotransferase: their inhibition modes and application to enzyme immunoassay.
Monoclonal and polyclonal antibodies against porcine mitochondrial aspartate aminotransferase (m-AST) were prepared in order to study their effect on the kinetics of the enzyme and their possible use as diagnostic reagents. The most stable hybridoma clone, designated MH-1, was selected and cultured for mass production of the monoclonal antibody MA-1. MA-1 was purified by affinity chromatography with m-AST as a ligand. The m-AST activity was inhibited uncompetitively by preincubation with MA-1, but preincubation with the polyclonal antibody uncompetitively by preincubation with MA-1, but preincubation with the polyclonal antibody raised in a rabbit resulted in noncompetitive inhibition of the enzyme. These results suggest the usefulness of a monoclonal antibody for studying the mechanism of catalysis. Sandwich enzyme immunoassay methods for m-AST using both polyclonal and monoclonal antibody-coated polystyrene balls were established and permitted the determination of porcine m-AST on the order of 10(-11) and 10(-10)M, respectively.
Suzuki
T
T
Kishi
Y
Y
Totani
M
M
Kagamiyama
H
H
Murachi
T
T
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001219:Aspartate Aminotransferases / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D007124:Immunoenzyme Techniques; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D008931:Mitochondria, Muscle / Q000201:enzymology*; D013552:Swine
D000906:Antibodies; D000911:Antibodies, Monoclonal; D001219:Aspartate Aminotransferases