A rapid radioimmunoassay for inducible rat submandibular gland cysteine proteinase inhibitor (cystatin).
A highly sensitive and specific radioimmunoassay for the determination of rat family 2 cystatins has been developed. Antibodies against cystatin were elicited in rabbits after coupling the protein to thyroglobulin. Antibodies were specific for rat family 2 cystatin and did not cross-react with human salivary cystatin, rat family 3 cystatin (kininogen) or other salivary proteins. Fifty percent precipitation of 125I-cystatin was obtained with a 1:100,000 dilution of antisera. Optimal conditions for each step of the assay were established from a series of initial experiments. Precipitation of the antigen-antibody complex was achieved with 10% polyethylene glycol in 0.05 M phosphate buffer, pH 7.5 in the presence of 0.1% gamma-globulin. A standard curve constructed by plotting the fraction of bound 125I-cystatin against submandibular gland cystatin in a logit-log mode yielded a straight line for cystatin concentrations between 0.2 and 200 ng. The lowest limit of detection of family 2 cystatin by RIA was 0.2 ng, and is 1000 fold more sensitive than enzyme-inhibition assays and Laurell rocket electrophoresis. Only trace amounts of cystatin were detected in submandibular, sublingual and parotid glands from normal rats. After isoproterenol-stimulation cystatin levels increased dramatically in salivary glands and moderately in kidney and pancreas.
Bedi
G S
GS
Department of Oral Biology, State University of New York, Buffalo 14214.
D000818:Animals; D000918:Antibody Specificity; D015891:Cystatins / Q000032:analysis* / Q000276:immunology; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D011919:Rats, Inbred Strains; D013363:Submandibular Gland / Q000032:analysis*
D015891:Cystatins; D007545:Isoproterenol