Use of protein G for preparation and characterization of rabbit antibodies against rat adipose tissue hormone-sensitive lipase.
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 micrograms 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.
D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins / Q000276:immunology*; D006728:Hormones / Q000494:pharmacology; D007074:Immunoglobulin G / Q000276:immunology*; D011817:Rabbits / Q000276:immunology; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D002787:Sterol Esterase / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins; D006728:Hormones; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D002787:Sterol Esterase