Proteomic Characterisation of Venom-Antivenom Interactions using Magnetic Bead-Based Immunoprecipitation and LC-MS/MS

Abstract

The diversity and complexity of venoms require a precise understanding of their interactions with antivenoms to identify which toxins are truly recognised and captured. This information is essential to assess the quality of available antivenoms, improve their production, and guide the development of next-generation antivenoms. In this context, it is important to have rapid, robust and reproducible in vitro methods capable of characterising these interactions in a reliable and directly actionable manner.

In this thesis, venom-antivenom interactions were studied in the Echis romani - EchiTabG pair using a magnetic bead-based immunocapture approach, coupled to LC-MS/MS and LC-IMS-MS/MS. Biolayer interferometry (BLI) was also used to monitor interactions in real time and to estimate the apparent affinity between Echis romani venom and the specific antivenom, EchiTabG, a monospecific antivenom composed of whole ovine IgG. Two experimental strategies based on magnetic beads were compared at different stages of the project: tosyl-activated beads enabling covalent immobilisation of antibodies, and protein G beads enabling oriented immobilisation via the Fc region of IgG. For the protein G strategy, two capture formats were evaluated: a solution pre-complexation format, where venom and antivenom are incubated together before bead capture, and a format where IgG are first immobilised on the beads, then incubated with the venom.

LC-MS/MS analyses highlighted a significant proportion of nonspecific capture with tosyl-activated beads, mainly associated with a background signal carried by PLA2s, consistent with nonspecific interactions between these proteins and the polystyrene surface of the beads. However, after exclusion of PLA2s during data processing, eluates obtained with EchiTabG showed toxin enrichment clearly higher than the controls, which supports the existence of a specific immunocapture. In contrast, immunocapture based on protein G beads showed little nonspecific signal, but the distribution of toxin families differed between the two capture formats. This difference probably reflects variations in complex formation in solution and in their recovery during capture via the Fc region. Based on these results, a bead saturation assay was set up by adding a “background” composed of proteins derived from Michigan Cancer Foundation-7 (MCF-7) cells, to reduce the nonspecific capture of PLA2s observed with tosyl-activated beads. Among the conditions tested, only the protein G strategy with prior antibody immobilisation showed behaviour compatible with saturation. BLI assays confirmed the specificity of the approach and showed binding dependent on venom concentration. Fitting a 1:1 binding model to this complex mixture enabled estimation of apparent values of k_on, k_off and K_D.

Ultimately, this work provides a solid methodological basis for the antivenomics of E. romani by showing that a magnetic bead immunocapture, validated and complemented by BLI, makes it possible to link toxin recognition coherently to measurable affinity parameters, paving the way for a finer evaluation of antivenoms.