Detection of anti-TNFa activity in canine hyperimmune serum using a TNFa inhibition assay

Abstract

Background: Increased serum tumor necrosis factor-α (TNFα) activity has been associated with onset of serious inflammatory diseases in dogs. Development of treatment with TNFα-antagonists has been limited by the unavailability of suitable reagents and potency assays for TNFα. Objectives: The objectives of this study were to optimize a cell-based assay to measure anti-TNFα activity in serum and plasma from hyperimmune (vaccinated with an Escherichia coli J5 bacterin) and unvaccinated canine donors; to use the assay to determine whether hyperimmune serum inhibits TNFα activity in vivo; and to determine whether soluble TNF receptor-1 (sTNFR1, a naturally occurring TNFα antagonist) contributes to anti-TNFα activity. Methods: Commercial plasma and serum from hyperimmune-frozen plasma (HFP) donors and unvaccinated fresh-frozen plasma (FFP) donors were used in the study. An L929-cell TNFα-inhibition assay (LTIA) was optimized to measure anti-TNFα activity. Using a rat subcutaneous pouch model of inflammation, the effects of HFP, FFP, a synthetic TNFα antagonist (Etanercept), and carprofen on TNFα activity were compared in vivo. Immunofluorescence was used to measure soluble sTNFR1 concentration. Results: Using the optimized LTIA, HFP serum but not FFP serum decreased canine TNFα activity (P<.01). HFP plasma and Etanercept (but not FFP plasma or carprofen) significantly decreased TNFα activity in pouch exudates (P<.05). A significantly higher concentration of sTNFR1 was found in HFP than FFP serum. Conclusions: Using the LTIA, anti-TNFα activity is readily measured in canine serum and inflammatory exudates. sTNFR1 appears to contribute to anti-TNFα activity in HFP serum. These results suggest HFP should be investigated further as a potential immunotherapeutic agent for controlling canine diseases in which TNFα is implicated.