Development and analytical validation of an enzyme-linked immunosorbent assay for the measurement of feline tumor necrosis factor α in serum.

Background: The role of tumor necrosis factor alpha (TNF-α), 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-α 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-α 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-α concentration in healthy cats was established. In addition, serum concentrations of fTNF-α in 39 cats with chronic enteropathies were compared with those in 20 healthy cats.

Results: The detection limit of the assay was 38.4 ng/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 < 38.4-223.5 ng/L. Serum concentrations of feline TNF-α 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-α in feline serum and should facilitate research into the importance of TNF-α in cats with chronic enteropathies.