Nucleotide sequence of equine erythropoietin and characterization of region-specific antibodies.

This research paper presents a study where the DNA sequence of equine erythropoietin (EPO) was identified and specific antibodies were developed that could differentiate between equine EPO and human EPO. This could help in identifying the abuse of recombinant human EPO in racehorses.

Methodology

• The researchers extracted RNA and lysate from the renal tissues of an adult Thoroughbred.
• A reverse transcriptase-polymerase chain reaction assay and a rapid amplification of cDNA ends method were used to determine the full-length cDNA of equine EPO.
• The inferred amino acid sequence was compared with erythropoietin sequences reported for other species.
• Four synthetic peptides were designed in two distinctive parts of the equine and human EPO amino acid sequences to generate antibodies specific for equine EPO and human EPO.
• The specificity of these antibodies was checked against a homogenized equine kidney supernatant and recombinant human EPO using western immunoblotting techniques.

Results

• Analysis of the 1,181 base pairs in the nucleotide sequence showed that equine EPO was a product of 192 amino acids.
• The compared amino acid sequences of EPO from other species showed a similarity of 81.0% to 90.6% with equine EPO.
• The developed antibodies were either specifically recognized by equine EPO or by recombinant human EPO molecules.
• The anti-human EPO (161 to 165) antibody specifically recognized recombinant human EPO. Conversely, the anti-equine EPO (133 to 144) antibody reacted with the equine kidney lysate.

Conclusions

• The study successfully identified the cDNA and amino acid sequence of equine EPO and developed region-specific antibodies that could distinctly recognize equine EPO or recombinant human EPO.
• The generated antibodies may contribute to distinguishing recombinant human EPO from equine EPO in tests devised to detect abuse of the former in racehorses.