The identification of nitrate reductase from Escherichia coli as the antigen for a monoclonal antibody of unknown specificity

The immunoaffinity chromatography of total membrane proteins from Escherich ia coli helped determine the specificity of the monoclonal antibody 3A6 tha t was obtained upon immunization of mice with nicotinamide nucleotide trans hydrogenase preparations and reacted with an unknown E. coli antigen. Prote ins with apparent molecular masses of 150, 45, and 20 kDa were isolated and identified by N-terminal sequencing as the subunits of nitrate reductase. This conclusion was confirmed by immunoblotting with the 3A6 antibody of th e proteins from the E. coil cells grown upon induction of nitrate reductase . It was shown that the 3A6 antibody specifically recognizes the a subunit of nitrate reductase, and the formation of the enzyme-antibody complex does not result in a loss of the enzyme catalytic activity.