Antivenomic approach of different Crotalus durissus collilineatus venoms

Abstract

Background: Our group has previously performed a proteomic study verifying that individual variations can occur among Crotalus durissus collilineatus venoms. These variations may lead to differences in venom toxicity and may result in lack of neutralization of some components by antivenom. In this way, this study aimed to evaluate the Brazilian anticrotalic serum capacity in recognizing twenty-two Crotalus durissus collilineatus venoms, as well as their fractions.

Methods: The indirect enzyme-linked immunosorbent assay (ELISA) was chosen to evaluate the efficacy of heterologous anticrotalic serum produced by Instituto Butantan (Brazil) in recognizing the twenty-two Crotalus durissus collilineatus venoms and the pool of them. Moreover, the venom pool was fractionated using reversed-phase fast protein liquid chromatography (RP-FPLC) and the obtained fractions were analyzed concerning antivenom recognition.

Results: Evaluation of venom variability by ELISA showed that all venom samples were recognized by the Brazilian anticrotalic antivenom. However, some particular venom fractions were poorly recognized.

Conclusion: This study demonstrated that the Brazilian anticrotalic serum recognizes all the different twenty-two venoms of C. d. collilineatus and their fractions, although in a quantitatively different way, which may impact the effectiveness of the antivenom therapy. These results confirm the need to use a pool of venoms with the greatest possible variability in the preparation of antivenoms, in order to improve their effectiveness.

Keywords: Antivenomic; Crotalic antivenom; Crotalus durissus collilineatus; Individual variation; Snake venoms.

Fig. 1

Antivenom recognition of C. d. collilineatus venoms and fractions performed by indirect enzyme-linked immunosorbent assay (ELISA) and chromatographic profiles of pooled venom. The 96-well plates were sensitized with 2 μg of (a) venoms (1–22) and (c) RP-FPLC fractions (0–44) diluted to 100 μL with carbonate-bicarbonate buffer (pH 9.6). The commercial anticrotalic serum (1,100) from Instituto Butantan was used to evaluate its capacity to recognize the venoms and their fractions using antihorse polyclonal antibodies peroxidase-labeled (1,3000) as secondary antibody. Positive control (C+): wells sensitized with anticrotalic antivenom (represented by horizontal dashed lines). Negative control (C-): non-sensitized wells. Absorbance reading was performed at 490 nm. Data are presented as mean ± SD, which were analyzed by ANOVA and Tukey’s multiple comparison test (quadruplicate assay). *p < 0.05, **p < 0.01 and ***p < 0.001 compared to C-; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 compared to C+. The arrow indicates crotamine-positive venom and fractions. (b) RP-FPLC of C. d. collilineatus pooled venom (22 mg) on a C18 column was carried out in a segmented concentration gradient from 6.3 to 100% of solution B (80% ACN in 0.1% TFA, represented by the blue dashed line) at a flow rate of 5 mL/min. Inset panel – whole chromatographic profile without magnification

Fig. 2

Densitometric analyses of the SDS-PAGE profiles of RP-FPLC fractions. (a) Fraction 0, (b) Fraction 19, (c) Fractions 21 and 22, (d) Fractions 35, 36, 37 and 39 and (e) Fraction 42. MW: molecular weight. Absorbance at 302 nm and the graphic created by software Image Lab™, version 5.2.1 (Bio-Rad Laboratories, Inc., California, USA)