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Updated: Jun 22, 2026
Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method
Published on: September 16, 2017
Detection of anti-TNFalpha activity in canine hyperimmune serum using a TNFalpha inhibition assay
Michael Kotiw1, Michael Morgan, Stephen M Taylor
1Department of Biological and Physical Sciences, Centre for Systems Biology, University of Southern Queensland, Toowoomba, Qld, Australia. kotiw@usq.edu.au
Background:
Increased serum tumor necrosis factor-alpha (TNFalpha) activity has been associated with onset of serious inflammatory diseases in dogs. Development of treatment with TNFalpha-antagonists has been limited by the unavailability of suitable reagents and potency assays for TNFalpha.
Objectives:
The objectives of this study were to optimize a cell-based assay to measure anti-TNFalpha 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 TNFalpha activity in vivo; and to determine whether soluble TNF receptor-1 (sTNFR1, a naturally occurring TNFalpha antagonist) contributes to anti-TNFalpha 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 TNFalpha-inhibition assay (LTIA) was optimized to measure anti-TNFalpha activity. Using a rat subcutaneous pouch model of inflammation, the effects of HFP, FFP, a synthetic TNFalpha antagonist (Etanercept), and carprofen on TNFalpha 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 TNFalpha activity (P<.01). HFP plasma and Etanercept (but not FFP plasma or carprofen) significantly decreased TNFalpha activity in pouch exudates (P<.05). A significantly higher concentration of sTNFR1 was found in HFP than FFP serum.
Conclusions:
Using the LTIA, anti-TNFalpha activity is readily measured in canine serum and inflammatory exudates. sTNFR1 appears to contribute to anti-TNFalpha activity in HFP serum. These results suggest HFP should be investigated further as a potential immunotherapeutic agent for controlling canine diseases in which TNFalpha is implicated.
