Fig. 3a shows strong similarities among the protein profiles of all Compound Library cell assay venoms. The presence of crotapotin, PLA2 and conjugated crotoxin was indicated by the similar mobility of the 10 kDa, 15 kDa and 30 kDa protein bands in the samples with the isolated crotoxin and PLA2 controls that were run in parallel. A band of 35 kDa, equivalent to gyroxin, could
be found in all the venom samples, although not in the purified fractions. Samples from the antivenom produced by the Instituto Butantan and samples of the Crotalus venoms were electrophoretically separated under reducing conditions on polyacrylamide gel electrophoresis (upper gel, 5%; lower gel, 12,5%). The protein bands were transferred to nitrocellulose membranes, treated with samples from the antisera (diluted 1:5000) and exposed to rabbit IgG anti-horse immunoglobulins as the second antibody. The recognition patterns of the plasma and antivenom from the Instituto Butantan were very similar, with the presence of bands near 15 kDa and 30 kDa, corresponding to PLA2 and crotoxin, respectively ( Fig. 3b and c). These proteins were detected in all the venoms with great intensity. Bands at 50 kDa and 60 kDa were also found in the C. d. terrificus, C. d. collilineatus and C. d. cascavella venoms, and a 10 kDa band, corresponding to
crotapotin, was detected in the C. d. collilineatus venom. In the plasma from Experimental Group 1, bands at 15 kDa and 30 kDa were observed for all the venoms, a 10 kDa GSK2118436 band was observed for the C. d. terrificus and C. d. collilineatus venoms, and a 60 kDa band was observed for the C. d. terrificus venom ( Fig. 3d). In the plasma from Experimental Group 2, bands at 15 kDa and 30 kDa were observed in all the venoms, a band at
10 kDa was observed for C. d. collilineatus venom, and bands at 50 kDa and 60 kDa were observed for the C. d. terrificus venom ( Fig. 3e). In the plasma from Experimental Group 3, only the 15 kDa band was observed for all the venoms ( Fig. 3f). Equal Fossariinae samples from the antivenoms were diluted (1:4.0 × 103 to 1:2.048 × 106) and assayed by ELISA. The obtained O.D. values at 492 nm were plotted against the corresponding serum dilutions, and dilutions giving O.D. values of 0.2 were used to calculate the number of U-ELISA/mL (Fig. 4). Antivenoms produced by the Instituto Butantan obtained the highest titers against the C. d. terrificus, C. d. collilineatus, C. d. cascavella and C. d. marajoensis venoms. Although no significant difference could be observed, there was a gap between the titers obtained against the crude venoms and those obtained against the purified components, suggesting that the high titers observed were related to the recognition of components other than crotoxin and PLA2. The titers obtained with plasma from Experimental Group 1 were the lowest against all the antigens tested. Plasma from Experimental Groups 2 and 3 showed high titers against the antigens tested.