Detection of anti‐TNFα activity in canine hyperimmune serum using a TNFα inhibition assay

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.