*4.1. Ethical Statement and Clinical Data Collection*

Patients included in the study (5) were hospitalized due to snakebite, at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), a reference hospital for snakebite treatment in Manaus, State of Amazonas, Brazil. Eligible patients were those diagnosed with clinical signs of envenomation by *Bothrops* snakes, which developed blisterings at the bitten limb during the time of hospital treatment. This study was approved by the Ethics Review Board of FMT-HVD (approval number CAAE 19380913.6.0000.5016/2013). All participants signed a consent form after an explanation of the study aims. Upon admission, epidemiological and clinical information were collected using a standardized questionnaire. Envenomation was classified as mild, moderate, or severe, according to

the Brazilian Ministry of Health guidelines [22]. The presence of pain, local bleeding, ecchymosis, necrosis, and systemic bleeding were also recorded. Patients were treated according to the Brazilian Ministry of Health protocols [22]. To collect the blister fluids, the area was cleaned using 2% chlorhexidine, and the fluid was collected using a sterile needle (13 × 4.5 mm) and a 1mL syringe. Subsequently, blister fluid was aliquoted and transferred to a sterile microtube and frozen at −80 ◦C until analysis.

### *4.2. ELISA Quantification of Antivenom and Venom in Blister Contents*

For antivenom detection, a 96-well plate was coated for 18 h at room temperature with 10 μg/mL of *B. atrox* venom in PBS (100 μL/well). The plates were then washed with PBS containing 0.05% TWEEN 20 solution and then blocked for 2 h at 37 ◦C with 300 μL/well of phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA). After blocking, the plates were washed and incubated for 2 h at 37 ◦C with the blister fluid 1000 times diluted in PBS containing 1% BSA and 0.05% TWEEN 20 or different dilutions of the standard curve, prepared with a known concentration of IgG (Fab'2 fragment) isolated from the same antivenom used for patient treatment. After a new washing step, peroxidase-labeled anti-horse IgG antibodies (SIGMA) were placed in each well, 2000 times diluted, and incubated at 37 ◦C for one hour. Antigen/antibody binding was detected by the addition of ortho-phenylenediamine. The antivenom concentration was calculated by linear regression of the standard curve. The same methodology was used for the detection of venom in the blister fluids, in which the plates were coated with Bothrops antivenom in a PBS buffer. After washing, the plates were incubated for 4 h at 37 ◦C with blister fluid (diluted 1:5 in PBS containing 1% BSA and 0.05% TWEEN 20) and the standard curve was prepared with *B. atrox* venom at known concentrations. The reaction was detected by rabbit serum anti-*B. atrox* venom followed by the addition of peroxidase labelled sheep anti-rabbit IgG (SIGMA) and ortho-phenylenediamine. The plates were read using a wavelength of 492 nm, and the venom concentration was calculated by linear regression of the standard curve. Experiments were carried out in triplicate and the results were expressed as mean ± S.E.M.

#### *4.3. Western Blot of Antivenom in Blister Contents*

The reactivity of blister contents with venom proteins was evaluated by Western blot. Briefly, *B. atrox* venom was electrophoresed under reducing conditions in 12% dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins contained in the gels were transferred to nitrocellulose membranes, which were blocked for 2 h at 37 ◦C with 500 μL of PBS containing 2% BSA (SIGMA-USA). Then, each membrane was incubated overnight at 4 ◦C with blister fluids from each patient, diluted at 1:2 in PBS containing 1% BSA. Following incubation, the membranes were washed with PBS containing 0.05% TWEEN. The capacity of the antivenom present in the blister content to bind to snake venom proteins immobilized on the nitrocellulose membranes was detected by chemiluminescence using peroxidase-labeled anti-horse antibody and ®Super Signal West Pico substrate from Thermo Scientific (Waltham, MA, USA). The images were captured after 15 s of membrane exposure. The positive control was Butantan commercial antivenom, similar to the one used to treat the patients, and the negative control was the plasma from a volunteer who never received antivenom as treatment.
