Generation of mouse anti-human urate anion exchanger antibody by genetic immunization and its identification.
BACKGROUND
Human urate anion exchanger (hURAT1) as a major urate transporter expressed on renal tubular epithelial cells regulates blood urate level by reabsorbing uric acid. Antibody is an important tool to study hURAT1. This study aimed, by genetic immunization, to produce mouse anti-hURAT1 polyclonal antibody with high throughput and high specificity and to detect the location of hURAT1 in human kidney.


METHODS
Human renal total RNA was isolated and the entire cDNA of hURAT1 was amplified by RT-PCR. The sequence of intracellular high antigenicity fragment (A280 to R349) was chosen by prediction software of protein antigenicity, and its cDNA was amplified from cDNA of hURAT1, and then cloned into pBQAP-TT vector to construct recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization. Mice were inoculated with this recombinant plasmid and two other adjuvant plasmids, pCMVi-GMCSF and pCMVi-Flt3L, which helped to enhance the antibody's generation. After four weeks, the mice were sacrificed to obtain the anti-hURAT1 antibody from serum. The antibody was identified by western blot analysis and immunohistochemistry. At the same time, rabbit anti-hURAT1 antibody was produced by protein immunization. The specificity and efficiency between the rabbit and mouse anti-hURAT1 antibody were compared by western blot analysis and immunohistochemistry.


RESULTS
The entire cDNA of hURAT1 and cDNA of its intracellular high immunogenic fragment were amplified successfully. Recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization was confirmed by restriction digestion and sequencing. Both the mouse anti-hURAT1 antibody and rabbit anti-hURAT1 antibody recognized 58 kD hURAT1 and 64 kD glycosylated hURAT1 protein bands in western blot. Immunohistochemically, hURAT1 was located at the brush border membrane of renal proximal tubular cells. In addition, the throughput and specificity of the mouse anti-hURAT1 antibody were higher than those of the rabbit anti-hURAT1 antibody.


CONCLUSIONS
Genetic immunization can generate anti-hURAT1 polyclonal antibody of high throughput and specificity.
Xu
Guo-shuang
GS
Department of Nephrology, Kidney Center and Key Lab of PLA, Chinese PLA General Hospital, Beijing 100853, China.
Wu
Di
D
Chen
Xiang-mei
XM
Shi
Suo-zhu
SZ
Hong
Quan
Q
Zhang
Ping
P
Lu
Yang
Y
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000276:immunology*; D005260:Female; D006801:Humans; D007114:Immunization; D007150:Immunohistochemistry; D007668:Kidney / Q000737:chemistry; D008297:Male; D051379:Mice; D027361:Organic Anion Transporters / Q000032:analysis / Q000276:immunology*; D027701:Organic Cation Transport Proteins; D010957:Plasmids; D011817:Rabbits
D000906:Antibodies; D002352:Carrier Proteins; D027361:Organic Anion Transporters; D027701:Organic Cation Transport Proteins; C491241:SLC22A12 protein, human