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. SAMPLE: Serum samples obtained from 6 healthy dogs and 3 dogs with pulmonary disease. PROCEDURES: 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 AND CLINICAL RELEVANCE: 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.