*4.2. Snake Venoms*

Lyophilized venoms (*Bothrops jararaca*, *B. alternatus*, *B. jararacussu*, *B. atrox amazonia* and *Bitis anetans*) were obtained from the Laboratory of Herpetology, Butantan Institute, São Paulo, Brazil, and stored at −20 ◦C. Venoms were resuspended in phosphate-buffered saline (PBS).

#### *4.3. Protein Quantification*

Protein concentration of the snake venom samples was determined by a microtiterbased Bradford Protein Assay (BioRad) in microplates using bovine serum albumin (BSA—Sigma–Aldrich, St. Louis, MO, USA) for the standard curve [25].

#### *4.4. Induction of Oral Tolerance and Immunization*

To establish the oral tolerance induction protocol, BALB/c mice were subdivided into 4 groups of animals (*n* = 3). Group I did not receive venom orally. Oral tolerance was induced by gavage administration of *B. jararaca* venom, and the mice received a single high dose of 1.8 mg (Group II) or 3 doses of 1 μg on the first, third and fifth day (Groups III and IV). At 12 days after the last low dose (Groups III and IV), all groups were intraperitoneally immunized with 1 μg of snake venom adsorbed to/encapsulated in nanostructured SBA-15 silica [26]. Groups I, II and III received *B. jararaca* venom, while Group IV was immunized with *B. atrox* venom. Antiserum corresponding to the 7th and 35th day after immunization was collected by bleeding from the retroorbital plexus and the antibody titer was determined by ELISA. Animals that did not undergo any procedure (*n* = 3) were considered the control group, and sera were added to the antibody titer assay.

Another oral tolerance induction protocol was then carried out. BALB/c mice were subdivided into 4 groups of animals (*n* = 5). Oral tolerance was induced by gavage administration of 1 μg of the *B. jararaca* venom on the first, third and fifth day (Groups I and III). Groups II and IV did not receive venom orally. Eight days after the last dose, mice were intraperitoneally immunized with 1 μg of snake venom adsorbed to/encapsulated in nanostructured SBA-15 silica. Groups I and II were immunized with *B. jararaca*, while Groups III and IV received *B. jararacussu* venom. Antiserum was collected by bleeding from the retroorbital plexus at 12, 25 and 45 days after immunization, and the antibody titers were determined by ELISA. Sera from mice of control group were also added. The crossreactivity of serum after induction of oral tolerance with *B. jararaca* venom was analyzed by Western blotting.

#### *4.5. ELISA Procedure*

IgG titer of mouse sera was determined by ELISA. *B. jararaca* or *B. jararacussu* venom was diluted to 10 μg/mL in carbonate buffer (50 mM Na2CO3/NaHCO3, pH 9.6), and 100 μL/well were added to a 96-well EIA/RIA Clear Flat Bottom Polystyrene High Bind Microplate (3590, Corning, NY, USA). The plate was incubated at 4 ◦C overnight, washed 3 times with PBS/0.05% Tween and then incubated with the PBS/5% BSA blocking solution at 37 ◦C for 2 h. The blocking solution was replaced with a serial twofold dilution of antiserum in PBS/1% BSA, starting at an appropriate dilution for each one. Sera from animals that did not receive any venom was used as negative control, and samples were incubated at 37 ◦C for 1 h. Wells were washed 5 times with PBS/0.05% Tween to remove unbound antibodies. HRP-conjugated anti-mouse IgG (H + L) (Promega, Madison, WI, USA) was used as secondary antibody at 1/2500 dilution, and incubation was at 37 ◦C for 1 h. The plate was washed again, and the substrate used was OPD (o-phenylenediamine) (Sigma– Aldrich) in phosphate-citrate buffer with H2O2. After 5 min incubation, the reaction was stopped by adding 0.2 M citric acid. The absorbance at 450 nm was measured with a microplate reader. Antibody titers were expressed as log2 maximum serum dilution giving a positive reaction at which the absorbance was equal to two times the control value of the control serum.

Immunoglobulin isotypes, IgG1 and IgG2a, were evaluated in mouse antisera. For this purpose, HRP rat anti-mouse IgG1 (BD Biosciences, San Jose, CA, USA) at 1/400 dilution and HRP rat anti-mouse IgG2a (BD Biosciences) at 1/1000 dilution were used as secondary antibody. Antibody titer was determined as described above.
