Development of high-specificity antibodies against renal urate transporters using genetic immunization

Abstract

Recently three proteins, playing central roles in the bi-directional transport of urate in renal proximal tubules, were identified: two members of the organic anion transporter (OAT) family, OAT1 and OAT3, and a protein that designated renal urate-anion exchanger (URAT1). Antibodies against these transporters are very important for investigating their expressions and functions. With the cytokine gene as a molecular adjuvant, genetic immunization-based antibody production offers several advantages including high specificity and high recognition to the native protein compared with current methods. We fused high antigenicity fragments of the three transporters to the plasmids pBQAP-TT containing T-cell epitopes and flanking regions from tetanus toxin, respectively. Gene gun immunization with these recombinant plasmids and two other adjuvant plasmids, which express granulocyte/macrophage colony-stimulating factor and FMS-like tyrosine kinase 3 ligand, induced high level immunoglobulin G antibodies, respectively. The native corresponding proteins of URAT1, OAT1 and OAT3, in human kidney can be recognized by their specific antibodies, respectively, with Western blot analysis and immunohistochemistry. Besides, URAT1 expression in Xenopus oocytes can also be recognized by its corresponding antibody with immuno-fluorescence. The successful production of the antibodies has provided an important tool for the study of UA transporters.