Detection and quantitation of a sodium-dependent sulfate cotransporter (NaSi-1) by sandwich-type enzyme-linked immunosorbent assay

The sodium-dependent sulfate transporter (NaSi-1) DNA has been recently ide ntified from rat kidney cortex. The objective of this study was to develop a quantitative assay for the NaSi-1 transporter protein. The NaSi-1 antigen was prepared by fusion protein techniques following analysis of the primar y sequence for antigenicity. Polyclonal and monoclonal antibodies against t he NaSi-1 antigen were raised in rabbits and mice, respectively. The specif icity of the raised antibodies was examined by Western analysis using brush -border membrane (BBM) and basolateral membrane (BLM) purified from rat kid ney cortex. Both NaSi-1 polyclonal and monoclonal antibodies detected a 69- kDa protein in the BBM. Using the purified monoclonal antibody as the captu re antibody and the polyclonal antibody as the detecting antibody, a simple and sensitive sandwich-type enzyme linked immunosorbent assay was develope d to quantitate NaSi-1 transporter protein levels in tissue. The specificit y of the assay was examined using BBM, BLM and NaSi-1-transfected Madin-Dar by canine kidney cells. The assay was capable of detecting NaSi-1 at levels as low as 6.58 fmol. The concentration of NaSi-1 transporter protein in cr ude membrane isolated from rat kidney cortex was 0.094+/-0.014 fmol/mu g pr otein (mean+/-SD of three preparations).