An ELISA for measuring tumour necrosis factor alpha in rat plasma

Tumour necrosis factor (TNF) is a cytokine with multiple biological activities which plays a pivotal role in the response of the body to infection. TNF is secreted in the the monomeric form and associates to yield a biologically active oligomeric molecule. Bioactive TNF can be measured in plasma using cytotoxic assays which employ the murine cell-lines L929 or WEHI 164 clone 13. However, it has become clear that inactive TNF also circulates in vivo, which is not detected by bioassay. These inactive forms may play an important role in the regulation of TNF activity. The rat is often used as a model for the study of acute infection and its systemic effects. Our aim was to develop an ELISA for rat TNF which would provide a convenient and cost effective method of assay. The assay employs two commercially available antibodies raised against recombinant murine TNF (rMuTNF) which exhibit cross-reactivity with rat TNF. The lower limit of detection for this assay was determined to be 39.0 pg/ml rMuTNF. The inter and intra-assay coefficients of variation were < 12.0%. Specificity of the assay was shown by the high degree of parallelism obtained between rMuTNF and commercially available rRatTNF. The assay described measures rat TNF in both plasma and tissue culture supernatant. The measurement of plasma concentrations of TNF using both the ELISA and bioassay may help elucidate more fully the biological importance of TNF.