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 versatility 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. Conventional approaches, such as HPLC analysis or autoradiography, are of limited sensitivity and resolution and have attendant biohazard risks. We now describe a versatile, facile and inexpensive means for quantifying and determining the distribution of BAPTA which exploits an immunological approach based on our generation of novel antibodies to BAPTA. Antibodies to BAPTA were prepared by immunizing rabbits with BAPTA conjugated to keyhole limpet hemocyanin via a zero-order cross-linking reagent-EDC. The ability of anti-BAPTA IgGs to recognize free or conjugated BAPTA was confirmed using enzyme-linked and immunoblotting assays made possible by our introduction of a BAPTA-BSA adduct. Using such assays, we show that the anti-BAPTA antibodies possess marked selectivity for BAPTA compared to several structurally-related BAPTA analogs. The utility of the anti-BAPTA antibodies in cell calcium research has been confirmed in two ways. First, by determining the spatial distribution 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 data demonstrate that anti-BAPTA antibodies are a powerful new tool with which to quantify BAPTA and to define the spatial and temporal distribution of this important calcium chelator in live cells. Such information should greatly aid the design of cell physiological experiments, the development of new chelators and the identification of sources of chelator selectivity in emerging therapeutic applications.
Tymianski
M
M
Playfair Neuroscience Unit, Toronto Hospital Research Institute, Ontario, Canada.
Sattler
R
R
Bernstein
G
G
Jones
O T
OT
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000888:Antibodies, Anti-Idiotypic; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006241:Haptens / Q000378:metabolism*; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007120:Immunochemistry; D007074:Immunoglobulin G / Q000276:immunology*; D011817:Rabbits / Q000276:immunology; D012710:Serum Albumin, Bovine / Q000276:immunology
D000906:Antibodies; D000888:Antibodies, Anti-Idiotypic; D002614:Chelating Agents; D006241:Haptens; D007106:Immune Sera; D007074:Immunoglobulin G; D012710:Serum Albumin, Bovine; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium