PREPARATION, CHARACTERIZATION AND UTILITY OF A NOVEL ANTIBODY FOR RESOLVING THE SPATIAL AND TEMPORAL DYNAMICS OF THE CALCIUM CHELATOR BAPTA

In spite of its importance as a tool to manipulate cell calcium, the v ersatility of the octadentate chelator BAPTA in cell physiological and diverse other applications is limited by the difficulty with which it can be quantified and its cell and tissue distributions determined. C onventional approaches, such as HPLC analysis or autoradiography, are of limited sensitivity and resolution and have attendant biohazard ris ks. We now describe a versatile, facile and inexpensive means for quan tifying and determining the distribution of BAPTA which exploits an im munological approach based on our generation of novel antibodies to BA PTA. Antibodies to BAPTA were prepared by immunizing rabbits with BAPT A conjugated to keyhole limpet hemocyanin via a zero-order cross-linki ng reagent -EDC. The ability of anti-BAPTA IgGs to recognize free or c onjugated BAPTA was confirmed using enzyme-linked and immunoblotting a ssays made possible by our introduction of a BAPTA-BSA adduct. Using s uch assays, we show that the anti-BAPTA antibodies possess marked sele ctivity for BAPTA compared to several structurally-related BAPTA analo gs. The utility of the anti-BAPTA antibodies in cell calcium research has been confirmed in two ways. First, by determining the spatial dist ribution of BAPTA through immunocytochemistry and confocal microscopy of cortical neurons loaded with BAPTA/AM and, second, by determination of the kinetics of loading and efflux of BAPTA through enzyme-linked cell immunoassays (ELISA) and immunocytochemistry. Together, these dat a demonstrate that anti-BAPTA antibodies are a powerful new tool with which to quantify BAPTA and to define the spatial and temporal distrib ution of this important calcium chelator in live cells. Such informati on should greatly aid the design of cell physiological experiments, th e development of new chelators and the identification of sources of ch elator selectivity in emerging therapeutic applications.