Monoclonal antibodies against ethacrynic acid high-sensitive Mg-ATPase.

Abstract

Ethacrynic acid (EA) high-sensitive Mg-ATPase activity is an anion sensitive, non-mitochondrial Mg-ATPase activity observed in the brain (Biochem. Pharmacol. 31, 877 and 4077, 1982). In the present study, we prepared monoclonal antibodies against EA high-sensitive Mg-ATPase for use in the purification of the enzyme and examination of its localization. BALB/c mouse was immunized with crude microsomal fractions from rat brain and boostered 4-7 weeks later. Antibody titer was measured by inhibition assay of EA high-sensitive Mg-ATPase activity. Hybridomas were obtained and cloned by the method of Köhler and Milstein. These selected hybridomas were injected i.p. to BALB/c mice, and the ascites were collected. Both of the hybridomas were positively stained by antimouse γ2b. or anti-mouse λ rabbit serum and FITC antirabbit immunoglobulins. IgG fractions of ascites were prepared by (NH4)2SO4. fractionation or by DEAE cellulose or protein A Sepharose 4B column chromatography. The antibodies inhibited EA high-sensitive Mg-ATPase activity without significant effects on activities of EA low-sensitive Mg-ATPase or Na, K-ATPase. These results suggest that monoclonal antibodies (IgG2b,λ) were obtained against the catalytic site of EA high-sensitive Mg-ATPase.