*4.9. ELISA Immunoassay*

Microplates of 96-well (Sarstedt, Germany) were sensitized with 100 μL/well of the heterologous proteins in the serial 1:2 dilution from 1 to 0.008 μg/mL and incubated in a humid chamber at 4 ◦C for 18 h. Crude venom and GST protein were used as controls under the same conditions. Subsequently, blocking was performed with PBS containing 1% bovine serum albumin (BSA) for 30 min. After blocking, the addition of 1:200 (7.5 μg) of polyclonal IgG anti-*C. iheringi* venom antibody diluted in PBS + 1% BSA at 37 ◦C for 1 h. Subsequently, the microplates were incubated with a peroxidase-conjugated anti-rabbit IgG antibody (1:5000) at 37 ◦C for 45 min. After this, a revelation solution of OPD (orthophenylenediamine) was added (1 mg of OPD, 2 mL of citrate/phosphate buffer, and 1 μL of hydrogen peroxide). Then, the microplates were statically incubated, in the dark, at 24 ◦C for 15 min, and sulfuric acid (H2SO4) 2 N was used to stop the reaction and the plate was read in an ELISA reader (Labsystems Multiskan, Thermo Fisher Scientific, Waltham, MA, USA) at 492 nm.

#### *4.10. Mice*

For the experiments, BALB/c male mice (between 18 and 20 g) were bred from the animal house facilities of the Butantan Institute, São Paulo, Brazil. The animals were kept in a controlled temperature, 12/12 light/dark cycle, and were provided with standard food and water *ad libitum*. The experimental protocols were approved by the Butantan Institute Ethical Committee for Animal Research (certified by CEUAIB n◦ 4300061120).
