title: Development of high-specificity antibodies against renal urate transporters using genetic immunization.
abstract: Recently three proteins, playing central roles in the bidirectional 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.
authors.affiliation: Department of Nephrology, Institute of Nephrology & Key Lab of PLA, General Hospital of PLA, Beijing 100853, P. R. China.
authors.forename: Guoshuang
authors.initials: G
authors.lastname: Xu
authors.forename: Xiangmei
authors.initials: X
authors.lastname: Chen
authors.forename: Di
authors.initials: D
authors.lastname: Wu
authors.forename: Suozhu
authors.initials: S
authors.lastname: Shi
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Wang
authors.forename: Rui
authors.initials: R
authors.lastname: Ding
authors.forename: Quan
authors.initials: Q
authors.lastname: Hong
authors.forename: Zhe
authors.initials: Z
authors.lastname: Feng
authors.forename: Shupeng
authors.initials: S
authors.lastname: Lin
authors.forename: Yang
authors.initials: Y
authors.lastname: Lu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D006801:Humans; D007114:Immunization; D007132:Immunoglobulin Isotypes / Q000032:analysis; D007668:Kidney / Q000737:chemistry*; D009865:Oocytes / Q000378:metabolism; D027361:Organic Anion Transporters / Q000235:genetics / Q000276:immunology*; D019444:Vaccines, DNA / Q000276:immunology*; D014981:Xenopus / Q000235:genetics
chemical_list: D000906:Antibodies; D007132:Immunoglobulin Isotypes; D027361:Organic Anion Transporters; D019444:Vaccines, DNA; C103862:urate transporter