1B). In addition, densitometric analysis of the SDS-PAGE was performed to estimate the purity of the mAb preparations. The purity grade for mAbs A85/9-4 (46%), 59/2-E4 (45%), and 6AD2-G5 (37%) is shown in Fig. 1C. The ability of purified mAbs to recognize ATM/ATR inhibitor the respective toxins by ELISA is shown in Fig. 2. The mAbs A85/9-4 and 59/2-E4, which recognize phospholipase A2 and Zn-metalloproteinase, respectively, were able to bind the antigen at the lowest concentration tested (10 ng/mL) at a relatively high optical density when compared to the control sample (Fig. 2A, B). However, mAb 6AD2-G5 was not as effective as the other two, as the final dilution that recognized the antigen was
8 μg/mL (Fig. 2C). In a previous study from our group, Petretski et al. (2000) showed that mAb 6AD2-G5 was very effective in neutralizing the catalytic activity of the thrombin-like enzyme and also it recognized a conformational epitope of the toxin. In fact, this could explain why mAb 6AD2-G5 weakly binds the target antigen adsorbed to the solid phase of the ELISA plate, as the adsorption of the antigen
to the plastic surface could result in slight changes in the antigen epitope structure. The neutralizing properties of the mAbs against their respective toxins are shown in Fig. 3. The ability of three different mAb 59/2-E4 concentrations to neutralize hemorrhage induced by 5 μg of venom is shown RO4929097 in Fig. 3A. Hemorrhage neutralization by the mAb 59/2-E4 was seen in a dose-dependent manner from 25 μg to 100 μg of antibody tested. Conversely, the ability of mAb 59/2-E4 to neutralize the enzyme’s catalytic activity was negligible (data not shown) when azocasein was used as substrate. This result indicates that mAb 59/2-E4 does not bind to the catalytic domain of B. atrox metalloproteinase.
The same pattern was observed for mAb MAJar 3 against jararhagin, a Bothrops jararaca PIII metalloproteinase ( Tanjoni et al., 2003). MAJar 3 efficiently neutralized the hemorrhagic activity of jararhagin without blocking the catalytic activity of the enzyme and was shown to bind to the C-terminal portion of the disintegrin domain, which could be in conformational proximity to the catalytic domain or functionally modulate Bay 11-7085 the hemorrhagic activity of the snake venom metalloproteinase. Because mAb 59/2-E4 neutralized the biological activity of hemorrhagin, which has properties similar to those of MAJar 3, it is possible that both mAbs recognize the same epitope. The myotoxic activity induced by PLA2 was inhibited when the enzyme was incubated with mAb A85/9-4 followed by injection into the gastrocnemius muscle of mice (Fig. 3B). The CK serum level was drastically reduced in mice treated with the specific mAb when compared to control mice, treated with the non-specific IgG.