A protocol to guide development of a sensitive ELISA for canine erythropoietin.
BACKGROUND
The determination of canine erythropoietin (EPO) concentration is crucial for monitoring the effect of human recombinant (hr) EPO therapy in dogs with chronic renal failure. Current assays are not specific for canine EPO and not sensitive enough to detect physiologic EPO levels in dogs.


OBJECTIVE
The objective of this study was to develop a simple and sensitive ELISA for canine EPO that could serve as a starting point for developing a commercially available assay.


METHODS
The ELISA was based on a mouse monoclonal antibody (mAb) and a rabbit polyclonal antibody (pAb) using 2 different immunization techniques: gene electrotransfer (GET) to generate the pAb and multiple antigen peptides (MAPs) to generate the mAb. The ELISA was performed using both EPO obtained from HeLa cells transfected with an expression plasmid encoding canine EPO and canine plasma with known concentrations of EPO.


RESULTS
The ELISA standard curve was linear for canine EPO concentrations of 7-66 mU/ml. Coefficients of variation were about 10%. No cross-reactivity between canine EPO and hrEPO was detected.


CONCLUSIONS
Using novel GET and MAP technology, we developed a sensitive and specific ELISA for canine EPO that can be used to guide future clinical applications for EPO detection and monitoring in dogs.
Giampaoli
Saverio
S
Istituto di Ricerche di Biologia Molecolare, Pomezia (Rome), Italy. saverio_giampaoli@merck.com
Facciabene
Andrea
A
Mennuni
Carmela
C
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D003429:Cross Reactions; D004283:Dog Diseases / Q000188:drug therapy; D004285:Dogs / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D004921:Erythropoietin / Q000097:blood* / Q000627:therapeutic use; D018014:Gene Transfer Techniques / Q000662:veterinary; D006367:HeLa Cells; D006801:Humans; D007676:Kidney Failure, Chronic / Q000188:drug therapy / Q000662:veterinary; D051379:Mice; D011817:Rabbits; D012680:Sensitivity and Specificity
D000906:Antibodies; D000911:Antibodies, Monoclonal; D004921:Erythropoietin