title: Development and validation of a sandwich ELISA for use in measuring concentrations of canine surfactant protein A in serum of dogs.
abstract: OBJECTIVE
  To develop and evaluate a sandwich ELISA incorporating rabbit antiserum specific for canine surfactant protein A (SP-A) for use in measuring concentrations of SP-A in serum of dogs.
  
  
  METHODS
  Serum samples obtained from 6 healthy dogs and 3 dogs with pulmonary disease.
  
  
  METHODS
  Rabbit antiserum was prepared against purified canine SP-A. The IgG fraction was isolated via protein G affinity chromatography and was then biotinylated. The sandwich ELISA was performed by use of anti-SP-A antibody (IgG) preabsorbed with sera from healthy dogs. Validity of the ELISA was confirmed by determination of the detection limit, precision, reproducibility, and accuracy. Serum SP-A concentrations were measured in 6 healthy dogs and 3 dogs with pulmonary disease.
  
  
  RESULTS
  Detection limit of the ELISA was 2.0 ng/mL. Within- and between-assay coefficients of variation ranged from 3.8% to 14.1% and from 15.5% to 35.6%, respectively. The observed-to-expected recovery ratio ranged from 77.1% to 89.9%. Serum SP-A concentrations measured by use of the ELISA were ≤ 2.3 ng/mL in the 6 healthy dogs, 25.6 ng/mL in a dog with severe cardiac pulmonary edema, 8.3 ng/mL in a dog with pneumonia, and 10.1 ng/mL in a dog with lung lobe torsion.
  
  
  CONCLUSIONS
  The sandwich ELISA was found to be useful for measuring purified canine SP-A concentrations and canine SP-A concentrations in serum samples. The ELISA was precise, reproducible, and accurate. The ELISA may be beneficial in assessing serum concentrations of canine SP-A as a potential biomarker of pulmonary diseases in dogs.
authors.affiliation: Department of Advanced Clinical Medicine, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, 598-8531, Japan. k-sone@land222.com
authors.forename: Katsuhito
authors.initials: K
authors.lastname: Sone
authors.forename: Hideo
authors.initials: H
authors.lastname: Akiyoshi
authors.forename: Mika
authors.initials: M
authors.lastname: Aoki
authors.forename: Shunji
authors.initials: S
authors.lastname: Sugii
authors.forename: Fumihito
authors.initials: F
authors.lastname: Ohashi
mesh_terms: D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D015415:Biomarkers / Q000097:blood; D004283:Dog Diseases / Q000175:diagnosis*; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D007074:Immunoglobulin G; D008171:Lung Diseases / Q000175:diagnosis / Q000662:veterinary*; D037662:Pulmonary Surfactant-Associated Protein A / Q000097:blood*; D011817:Rabbits
chemical_list: D000888:Antibodies, Anti-Idiotypic; D015415:Biomarkers; D007074:Immunoglobulin G; D037662:Pulmonary Surfactant-Associated Protein A