A Simplified Method for the Estimation of Glutathione Peroxidase Activity and Selenium Concentration in Bovine Blood

Abstract

Twelve hybridoma clones were established that produced mouse monoclonal antibodies to bovine erythrocyte glutathione peroxidase. In these monoclonal antibodies, GPF-1, GPI-2, and GPJ-1, which showed marked reaction to this enzyme, were examined for reactivity to erythrocyte lysates from 12 different species of animals and from humans and for the inhibition of glutathione peroxidase activity. GPF-1 and GPJ-1 reacted markedly with glutathione peroxidase in erythrocyte lysates from ruminants and pigs and inhibited enzymatic activity. Conversely, GPI-2 showed positive reaction to hemolysate from all mammals used, except for mice, and did not inhibit enzymatic activity. To determine the concentration of bovine glutathione peroxidase in erythrocyte lysates, a sandwich ELISA was developed using GPJ-1, both as a coated antibody and as a peroxidase-labeled antibody. This ELISA system was a sensitive procedure with a detection limit of 6 ng/ml for glutathione peroxidase protein. Using blood samples from 121 cows, optical density by this ELISA was well correlated with the glutathione peroxidase activity and with the selenium concentration in bovine whole blood. The sandwich ELISA using GPJ-1 is a rapid and simple screening method to estimate glutathione peroxidase activity and selenium concentration in bovine whole blood.