title: A protocol to guide development of a sensitive ELISA for canine erythropoietin.
abstract: 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.
authors.affiliation: Istituto di Ricerche di Biologia Molecolare, Pomezia (Rome), Italy. saverio_giampaoli@merck.com
authors.forename: Saverio
authors.initials: S
authors.lastname: Giampaoli
authors.forename: Andrea
authors.initials: A
authors.lastname: Facciabene
authors.forename: Carmela
authors.initials: C
authors.lastname: Mennuni
mesh_terms: 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
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D004921:Erythropoietin