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 identified 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 primary sequence for antigenicity. Polyclonal and monoclonal antibodies against the NaSi-1 antigen were raised in rabbits and mice, respectively. The specificity of the raised antibodies was examined by Western analysis using brush-border membrane (BBM) and basolateral membrane (BLM) purified from rat kidney cortex. Both NaSi-1 polyclonal and monoclonal antibodies detected a 69-kDa protein in the BBM. Using the purified monoclonal antibody as the capture antibody and the polyclonal antibody as the detecting antibody, a simple and sensitive sandwich-type enzyme-linked immunosorbent assay was developed to quantitate NaSi-1 transporter protein levels in tissue. The specificity of the assay was examined using BBM, BLM and NaSi-1-transfected Madin-Darby 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 crude membrane isolated from rat kidney cortex was 0.094+/-0.014 fmol/ microg protein (mean+/-SD of three preparations).
Sagawa
K
K
Department of Pharmaceutics, State University of New York at Buffalo, Amherst NY 14260, USA.
DuBois
D C
DC
Han
B
B
Almon
R R
RR
Biber
J
J
Murer
H
H
Morris
M E
ME
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D027682:Cation Transport Proteins; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007668:Kidney; D007672:Kidney Cortex / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D000077003:Sodium Sulfate Cotransporter; D027981:Symporters
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D011993:Recombinant Fusion Proteins; D000077003:Sodium Sulfate Cotransporter; D027981:Symporters