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

Fumio Sato Equine Research Institute, Japan Racing Association, 321-4 Tokami, Utsunomiya 320-0856, Japan.

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Shozo Yamashita Laboratory of Racing Chemistry, 1731-2 Tsuruta, Utsunomiya 320-0851, Japan.

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Takaharu Kugo Equine Research Institute, Japan Racing Association, 321-4 Tokami, Utsunomiya 320-0856, Japan.

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Telhisa Hasegawa Equine Research Institute, Japan Racing Association, 321-4 Tokami, Utsunomiya 320-0856, Japan.

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Isao Mitsui Laboratory of Racing Chemistry, 1731-2 Tsuruta, Utsunomiya 320-0851, Japan.

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Isao Kijima-Suda Laboratory of Racing Chemistry, 1731-2 Tsuruta, Utsunomiya 320-0851, Japan.

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Abstract

Objective—To determine the full-length complementary DNA (cDNA) sequence of equine erythropoietin (EPO) and to develop region-specific antibodies to differentiate equine EPO (eEPO) and human EPO (hEPO).

Sample Population—RNA and lysate extracted from renal tissues of an adult Thoroughbred.

Procedure—Full-length cDNA was determined by use of a reverse transcriptase-polymerase chain reaction assay and a rapid amplification of cDNA ends method. The deduced amino acid sequence was compared with sequences of EPO reported for other species. Furthermore, 4 synthetic peptides were designed in 2 distinctive parts of the eEPO and hEPO amino acid sequences to obtain antibodies specific for eEPO and hEPO. Specificity of the antibodies was tested against supernatant of homogenized equine kidney and recombinant hEPO (rhEPO) by use of western immunoblotting techniques.

Results—Analysis of the 1,181 bp in the nucleotide sequence revealed that eEPO was a residue of 192 amino acids. Similarity of eEPO with amino acid sequences of EPO from other species was 81.0% to 90.6%. Antibodies were specifically recognized by eEPO or rhEPO molecules. Anti-hEPO (161 to 165) antibody specifically recognized rhEPO. In contrast, anti-eEPO (133 to 144) antibody reacted with the equine kidney lysate.

Conclusions and Clinical Relevance—We determined the cDNA and amino acid sequence of eEPO and developed region-specific antibodies that specifically recognized eEPO or rhEPO. These antibodies may be useful in distinguishing rhEPO from eEPO in a test to detect the misuse of rhEPO in racehorses. ( Am J Vet Res2004;65:15–19)

Abstract

Objective—To determine the full-length complementary DNA (cDNA) sequence of equine erythropoietin (EPO) and to develop region-specific antibodies to differentiate equine EPO (eEPO) and human EPO (hEPO).

Sample Population—RNA and lysate extracted from renal tissues of an adult Thoroughbred.

Procedure—Full-length cDNA was determined by use of a reverse transcriptase-polymerase chain reaction assay and a rapid amplification of cDNA ends method. The deduced amino acid sequence was compared with sequences of EPO reported for other species. Furthermore, 4 synthetic peptides were designed in 2 distinctive parts of the eEPO and hEPO amino acid sequences to obtain antibodies specific for eEPO and hEPO. Specificity of the antibodies was tested against supernatant of homogenized equine kidney and recombinant hEPO (rhEPO) by use of western immunoblotting techniques.

Results—Analysis of the 1,181 bp in the nucleotide sequence revealed that eEPO was a residue of 192 amino acids. Similarity of eEPO with amino acid sequences of EPO from other species was 81.0% to 90.6%. Antibodies were specifically recognized by eEPO or rhEPO molecules. Anti-hEPO (161 to 165) antibody specifically recognized rhEPO. In contrast, anti-eEPO (133 to 144) antibody reacted with the equine kidney lysate.

Conclusions and Clinical Relevance—We determined the cDNA and amino acid sequence of eEPO and developed region-specific antibodies that specifically recognized eEPO or rhEPO. These antibodies may be useful in distinguishing rhEPO from eEPO in a test to detect the misuse of rhEPO in racehorses. ( Am J Vet Res2004;65:15–19)

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