In mammals, the neuropeptide nerve growth factor (NGF) plays an important role in pain signalling by increasing the sensitivity of sensory nerve fibres. As NGF is released as a response to noxious stimuli, elevated blood concentrations were found to be present in the osteoarthritic joints of dogs. Therapeutic antibodies specifically neutralizing NGF therefore represent effective novel therapeutic means to reduce acute and chronic pain in different animal species by blocking the interaction between NGF and its receptor. With bedinvetmab (Librela, Zoetis), a fully canine monoclonal anti-NGF antibody has already received clinical approval in both the US and EU. Related substances such as the caninised anti-NGF monoclonal antibody ranevetmab are available for research purposes and therefore also for doping in animal sports.
Since analgesics are among the prohibited substances in dog sports competitions, the aim of this research project was to develop a specific and sensitive detection assay for anti-NGF antibodies such as bedinvetmab and ranevetmab by using immunoaffinity purification with NGF-conjugated magnetic beads for sample extraction, tryptic digestion, and LC-HRMS/MS. The method was comprehensively characterized and found to be highly sensitive and specific. It can readily be modified to include other therapeutic antibodies potentially relevant in canine sports drug testing by using additional ligands for immunoaffinity purification and is also easily transferable between species.