Guoshuang Xu1, An Liu, Xiaowei Liu. 1. Department of Nephrology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, PR China. xugsh882003@yahoo.com.cn
Abstract
BACKGROUND: Rat dicarboxylate transporter (SDCT1), expressed in renal tubular epithelial cells, plays a key role in regulating blood and urinary citrate level by reabsorbing citrate from the lumen. Antibodies against this transporter are very important for investigating its expression and function. With the cytokine gene as a molecular adjuvant, genetic immunization-based antibody production offers several advantages compared with current methods. This study aimed, by genetic immunization, to produce a high-specificity antibody against SDCT1. METHODS: We fused a high-antigenicity fragment of SDCT1 to the plasmid pBQAP-TT containing T-cell epitopes and flanking regions from tetanus toxin. Mice were immunized by gene-gun immunization with recombinant plasmid and two other adjuvant plasmids that express granulocyte/macrophage colony-stimulating factor and FMS-like tyrosine kinase 3 ligand, respectively. The titer of the antibody was detected by enzyme-linked immunosorbent assay (ELISA). Specificity of the antibody was identified with SDCT1 native protein in rat kidney by Western blot analysis and immunohistochemistry, and with SDCT1 protein expressed on Xenopus oocytes plasma membranes by immunofluorescence. RESULTS: ELISA measurements showed that the antibody titer was 1:32,000. The native protein of SDCT1 in rat kidney can be recognized by this antibody with Western blot analysis and immunohistochemistry. Immunofluorescence showed that this antibody also recognized SDCT1 protein targeted to Xenopus oocytes plasma membranes into which SDCT1 full-length cRNA was injected. CONCLUSION: Generation of a high-specificity immunoglobulin G antibody against SDCT1 by genetic immunization has provided an important tool for the study of citrate transport.
BACKGROUND:Rat dicarboxylate transporter (SDCT1), expressed in renal tubular epithelial cells, plays a key role in regulating blood and urinary citrate level by reabsorbing citrate from the lumen. Antibodies against this transporter are very important for investigating its expression and function. With the cytokine gene as a molecular adjuvant, genetic immunization-based antibody production offers several advantages compared with current methods. This study aimed, by genetic immunization, to produce a high-specificity antibody against SDCT1. METHODS: We fused a high-antigenicity fragment of SDCT1 to the plasmid pBQAP-TT containing T-cell epitopes and flanking regions from tetanus toxin. Mice were immunized by gene-gun immunization with recombinant plasmid and two other adjuvant plasmids that express granulocyte/macrophage colony-stimulating factor and FMS-like tyrosine kinase 3 ligand, respectively. The titer of the antibody was detected by enzyme-linked immunosorbent assay (ELISA). Specificity of the antibody was identified with SDCT1 native protein in rat kidney by Western blot analysis and immunohistochemistry, and with SDCT1 protein expressed on Xenopus oocytes plasma membranes by immunofluorescence. RESULTS: ELISA measurements showed that the antibody titer was 1:32,000. The native protein of SDCT1 in rat kidney can be recognized by this antibody with Western blot analysis and immunohistochemistry. Immunofluorescence showed that this antibody also recognized SDCT1 protein targeted to Xenopus oocytes plasma membranes into which SDCT1 full-length cRNA was injected. CONCLUSION: Generation of a high-specificity immunoglobulin G antibody against SDCT1 by genetic immunization has provided an important tool for the study of citrate transport.