Identification of an extracellular epitope of the Na⁺/Ca²⁺ exchanger (NCX1) using monoclonal antibodies

We generated a panel of monoclonal antibodies (mAbs) that bind the N-terminus of the Na⁺/Ca²⁺ exchanger (NCX1). The epitope recognized by these antibodies is highly conserved across species. Six mAbs, of different IgG subclasses, recognize amino acids one through forty of the mature protein, and have K_d values ranging from 0.7 to 5.0 nM. The mAbs were raised against rabbit and bovine fusion proteins, and characterized by ELISA, Western analysis, immunoprecipitation, and immunofluorescence analysis (IFA). Western analysis of mouse heart, brain, kidney, lung, spleen, liver, and skeletal muscle detects two well recognized NCX1-related proteins of 160- and 120-kDa. Immunoprecipitation from rabbit kidney and a monkey kidney cell line. LLC-MK₂, also detects the same two NCX1-related proteins. IFA reveals labeling of the basolateral membrane of both rabbit kidney distal tubule and LLC-MK₂ cells. The distal tubular labeling pattern (majority of cells in the connecting tubule) is consistent with previous reports of NCX1 localization in kidney. Some of the mAbs react with the native form of NCX1 as it is expressed on live LLC-MK₂ cells, indicating that their epitope is extracellular, mAb binding to an extracellular epitope should allow for their use as tools in immunodissection and fluorescent activated cell sorting. The species and tissue-specific isoform cross-reactivity of the antibodies, as well as their applicability to multiple immunoassays, will make them important tools for future studies on the regulation of NCX1.