Preliminary development of monoclonal antibody-based ELISA assays for Ixodes holocyclus paralysis tick holocyclotoxin assessments as an alternative to in vivo approaches.

Tick paralysis in companion animal pets is primarily caused by holocyclotoxins secreted by the salivary glands of the Australian eastern paralysis tick, Ixodes holocyclus. These toxins have been traditionally identified and quantified using in vivo neonate assays that are relatively reproducible but constrained by animal ethics. Thus improved analytical methods are needed to support the standardisation of antiserum products, toxin characterisation, and the development of next-generation therapeutics to treat companion animal paralysis. Specific anti-holocyclotoxin (anti-HT) monoclonal antibodies were produced against predicted B cell epitopes from HT-1 and HT-5. The monoclonal antibodies were characterised using surface plasmon resonance kinetics and used to develop ELISAs. The sensitivity, reproducibility, and specificity of the monoclonal antibody-based ELISAs for the detection of HT-1 and HT-5 are high. The results from the Surface Plasmon Resonance (SPR) kinetics showed that the selected antibodies have high affinities for the these HTs. The optimised ELISAs enabled high-affinity HT-1 detection across a 1- 60 ng/mL range and distinguished toxin-neutralising capacity among commercial anti-tick sera. Finally, this immunoassay, which utilises monoclonal antibodies against HT-1 and HT-5, represents a major step forward for the quantification of HTs and provides a much more ethical alternative to in vivo testing. Its application in the assessment of tick antisera makes this technique highly relevant and provides potential for the development of recombinant anti-toxin treatments. Future research should focus on developing a panel of mAbs to detect all 19 HT types.