Development and analytical validation of an enzyme-linked immunosorbent assay for the measurement of feline tumor necrosis factor in serum.
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abstract
BACKGROUND: The role of tumor necrosis factor alpha (TNF-), a cytokine shown to play a crucial role in human Crohn's disease patients, has not been documented in cats with chronic enteropathies. Also, currently, no validated assay for measurement of TNF- in cats is available. OBJECTIVES: The objective of this study was to develop and analytically validate an enzyme-linked immunosorbent assay (ELISA) for the quantification of TNF- in serum from cats. METHODS: A sandwich ELISA was developed and analytically validated by assessment of detection limit, linearity, accuracy, precision, and reproducibility. A control range for serum fTNF- concentration in healthy cats was established. In addition, serum concentrations of fTNF- in 39 cats with chronic enteropathies were compared with those in 20 healthy cats. RESULTS: The detection limit of the assay was 38.4ng/L. Observed-to-expected ratios for serial dilutions of 4 serum samples ranged from 75.1% to 111.9%. Observed-to-expected ratios for spiking recovery for 4 serum samples ranged from 91.3% to 129.7%. Coefficients of variation for intra- and inter-assay variability ranged from 3.9% to 7.6% and from 7.8% to 12.5%, respectively. The control range of the assay was <38.4-223.5ng/L. Serum concentrations of feline TNF- were significantly higher in cats with chronic enteropathies and diarrhea than in cats with chronic enteropathies without diarrhea, or in healthy control cats. CONCLUSIONS: The ELISA described here was suitable for the quantification of fTNF- in feline serum and should facilitate research into the importance of TNF- in cats with chronic enteropathies.
