1% BSA for 1 h at room temperature on a horizontal shaker. After being washed three times with PBS plus 0.05% Tween-20, the membranes were incubated with rabbit anti-horse IgG conjugated to alkaline phosphatase (whole molecule) diluted 1:7500 in PBS plus 0.1% BSA and 0.05% Tween-20. Then, the membranes were incubated for 1 h at room temperature on a horizontal shaker. The membranes were washed three times with PBS plus 0.05% Tween-20 and placed in developing solution for Western blotting.

The reaction was terminated by washing with distilled water. Polystyrene, high-affinity ELISA plates (96 wells) were coated with 1.0 μg of crude C. d. terrificus, C. d. collilineatus, C. d. cascavella or C. d. marajoensis venom in 100 μL of PBS buffer and kept overnight at 4 °C. In some assays, crotoxin or PLA2 purified from C. d. terrificus was used as the antigen. The wells were

blocked for 2 h at 37 °C Selleckchem PLX4032 with 200 μL of PBS plus 5% BSA. The wells were washed with 200 uL Pexidartinib clinical trial of PBS. Serial dilutions of horse IgG or F(ab′)2 preparations (1:4000 to 2,048,000) in PBS plus 0.1% BSA were prepared, and 100 μL of each dilution was added to individual wells. The plates were then incubated at 37 °C for 1 h, and then, the wells were washed three times with the wash buffer. Rabbit peroxidase-conjugated anti-horse IgG (whole molecule) (Sigma Aldrich, St. Louis, MO) diluted (1:20,000) in PBS plus 0.1% BSA and 0.05% Tween-20 (100 μL/well) was added to the plates. The plates were incubated for 1 h at 37 °C. After three washes with the wash buffer, 50 μL of substrate buffer were added to each well, and plates were incubated at room temperature for Dichloromethane dehalogenase 15 min. The reaction was terminated with 50 μL of 4 N sulfuric acid per well. Absorbance was recorded at 492 nm using an ELISA plate reader (Labsystems Multiskan Ex, Thermo Fisher Scientific Inc., Walthan, MA). IgG from horses collected before immunization was always used as a negative control. The IgG dilution giving an optical density of 0.2 was used to calculate the U-ELISA per milliliter of the undiluted IgG solution. One U-ELISA was defined as

the smallest dilution of antibody that presented an O.D. of 0.2 under conditions of the ELISA assay, as described previously ( Almeida et al., 2008). The value was then multiplied by 10 to correspond to milliliters. The affinity of anti-Crotalus antibody was measured by ELISA, as described above, with the inclusion of a potassium thiocyanate (KSCN) elution step ( Pullen et al., 1986; Romero-Steiner et al., 2005). After the serum incubation step, dilutions of KSCN (0.0–5.0 M, in intervals of 0.50 M) in PBS were added to the wells and incubated for 30 min at room temperature. The remaining bound antibodies were detected with rabbit peroxidase-conjugated anti-horse IgG (whole molecule) (Sigma Aldrich, St. Louis, MO) diluted (1:20,000) in PBS plus 0.