Salivary glands synthesize and secrete cyclic AMP-reactive proteins (cARP)-regulatory (R) subunits of cyclic AMP (cAMP)-dependent protein kinase. The R subunits are generally intracellular eukaryotic cARP which regulate protein phosphorylation by formation or dissociation of the holoenzyme. Recent animal studies have shown that cARP (mainly the type Il isoform) are present in saliva and in other body fluids and may have separate extracellular function. The purpose of this study is to prepare and test specific monoclonal antibodies (mAb) using cARP purified from human saliva as antigen (Ag). Whole saliva and parotid fluid were collected from a population of healthy volunteers without diagnosed disease or oral pathology. Salivary cARP activity was measured using photoaffinity labeling with an azido analog of AMP, separating proteins by electrophoresis and identifying cARP by autoradiography. Densitometric analyses of the radioactive bands from saliva of 12 male and 11 female volunteers showed that each sample of parotid and whole saliva contained cARP (in a ratio of about 10:1) with the relative mobility of RII compared to standards, providing a sufficient quantity of Ag for immunization (approximately 0.1 to 1.0 ug/ml). Isolation of cARP will be by affinity chromatography using cAMP-Sepharose which binds only R subunits. The cARP will be eluted, collected, checked for purity by photoaffinity labeling and electrophoresis, concentrated and used in immunizations. Antibodies to human salivary cARP will be raised in mice to produce antibody secreting cells for fusion to form hybridomas which secrete MAb. Limiting dilution clonal selection and characterization of MAb will be carried out using ELlSA, immunodiffusion. Western blotting and immunocytochemistry to ensure that the optimal testing methods hake been selected. As in other tissues, cARP in oral epithelium is likely to mediate cellular signal processing and secretory cARP in saliva may be used as an index of systemic, hormone- dependent, or drug-induced disorders. The MAb will be used to determine baseline values of cARP in human saliva of a population of normal subjects and to immunocytochemically localize cAPK subunits in glandular epithelium of rat parotid, exorbital lacrimal and pancreatic gland tissue as well as in ameloblasts involved in secretion of rat incisor enamel. Additionally, these Ab will be used for the intracellular localization and quantitation of cARP in oral tissues (archival samples of human tissue) and in saliva of patients with diagnosed periodontitis. Our preliminary findings showed unique, individual cARP pleiomorphy. These reagents may also be commercially developed for application in clinical or forensic science.
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