Optimization of a procedure to accurately detect equine TNFα in serum samples

Abstract

The systemic component of chronic inflammatory diseases may lead to clinical complications. High levels of TNFα, a pro-inflammatory cytokine, are found in human patients with COPD and asthma. Horses are also susceptible to an array of chronic inflammatory disorders possibly associated with systemic inflammation, including respiratory diseases. Currently, there is no commercially available ELISA validated to assess TNFα in equine serum samples. Moreover, the reported normal mean concentration of serum TNFα in horses vary greatly. Hence, we sought to optimize and validate a procedure to quantify this cytokine in equine serum samples using a sandwich ELISA. Our results indicate that the nature of diluent buffers greatly impact the detection of TNFα in equine serum samples as its quantification increased in some cases from non-detectable levels to the ng/ml range. Linearity assays performed with serum samples from six animals serially diluted in four different buffers showed that serum matrix interference was animal-dependant. The specificity of TNFα detection was also assessed. Our optimized assay conditions were validated by quantifying levels of TNFα in serum samples from normal horses and horses affected with chronic pulmonary disease (heaves).