Use of protein G for preparation and characterization of rabbit antibodies against rat adipose tissue hormone-sensitive lipase

Abstract

The newly described immunoglobulin G-binding streptococcal surface protein, protein G, was used to prepare and characterize rabbit antibodies. The antibodies were directed against rat hormone-sensitive lipase, the rate-limiting enzyme in the hydrolysis of the triacylglycerols stored in adipose tissue. Antiserum was obtained after two injections with 20 μg enzyme protein, and the immunoglobulin fraction was obtained using a protein G-based solid-phase radioimmunoassay. The hydrolysis of acylglycerols by the enzyme was inhibited by the antibodies, and the enzyme could be efficiently removed from a solution using the antibodies and heat-killed streptococci expressing surface protein G. By Western blot and detection with 125I-protein G, the antibodies were found to selectively bind to hormone-sensitive lipase and to a smaller extent to two minor contaminants, possibly proteolytic fragments of the lipase. The amount of 125I-labelled protein G bound to the lipase on the blot was quantitatively related to the amount of enzyme protein down to the detection limit 10 ng.