Fig. 3
Antivenomic analytical strategies. A schematic representation of immunological approaches that have been combined with proteomic analysis of snake venoms, aiming to assess the immunorecognition of venom components by antibodies present in a given antivenom. a Immunoblotting, performed on electrotransferred membranes from two-dimensional gel electrophoresis (2DE) venom separations, identifies spots that are immunorecognized by the antivenom, in an essentially qualitative way. Immunoblotting can also be performed on membranes from the electrophoresis step (second dimension separation by SDS-PAGE) of the snake venomics strategy (see text and Fig. 1c). b ‘First generation’ antivenomics evaluates the immunodepletion of venom components after addition of antivenom and removal of precipitated immunocomplexes. The remaining supernatant is analyzed by HPLC and its profile is compared to that of a control venom aliquot. Differences in the chromatographic peaks between the antivenom-treated venom and the control venom can be quantified by integration of their peak areas, representing the immunodepletion of recognized components. c ‘Second generation’ antivenomics evaluates the venom components that are captured by an antivenom that has been covalently linked to beads, following the principles of immunoaffinity chromatography. Whole venom is incubated with this matrix and the unbound components are collected. After washing out the non-binding venom components, a change in pH elutes the bound venom fraction. Both samples are finally analyzed by HPLC, and their profiles are compared to that of a control sample of venom. Quantitative estimations of the degree of immunorecognition of components are performed as described for panel b by integration of chromatographic peak areas [58]. d HPLC/ELISA-based assessment of immunorecognition of venom components by an antivenom, or HPLC/ELISA-based immunoprofiling, is performed by coating microwell plates with a normalized amount of venom fractions obtained from the HPLC profile of the venom. Then, antivenom is added to each well and the bound antibodies (Ab) are detected by conventional ELISA