A Rapid Radioimmunoassay for Inducible Rat Submandibular Gland Cysteine Proteinase Inhibitor (Cystatin)

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Abstract
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 0.05M phosphate buffer, pH 7.5 in the presence of 0.1% γ-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.