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International Urology and Nephrology

Erschienen in:

01.03.2009 | Nephrology - Original Paper

Use of genetic immunization to generate a high-level antibody against rat dicarboxylate transporter

verfasst von: Guoshuang Xu, An Liu, Xiaowei Liu

Erschienen in: International Urology and Nephrology | Ausgabe 1/2009

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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.

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Titel: Use of genetic immunization to generate a high-level antibody against rat dicarboxylate transporter
verfasst von: Guoshuang Xu
An Liu
Xiaowei Liu
Publikationsdatum: 01.03.2009
Verlag: Springer Netherlands
Erschienen in: International Urology and Nephrology / Ausgabe 1/2009
Print ISSN: 0301-1623
Elektronische ISSN: 1573-2584
DOI: https://doi.org/10.1007/s11255-008-9432-x

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Abstract

Background

Methods

Results

Conclusion

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