Comparación de la especificidad y capacidad neutralizante de dos antivenenos antiofídicos retados con veneno de serpiente del género Bothrops de Colombia

Introduction. In Colombia the antivenom against snakebite from the National Institute of Heatlh (INS) and the antivenom from the Bioclon Intitute, belonging to Laboratorios Silane of Mexico, Antivipmyn-Tri (AVP-T), but neither includes venom of Bothrops rhombeatus species for their production. Objective. The aim of this research was to evaluate the neutralizing capacity, specificity and affinity of INS and AVP-T antivenoms with B. rhombeatus venom, using the Bothrops asper and Bothrops atrox venom as a reference and control for the experiments. Methods. The research was carried out in the Instrumental Analysis Laboratory of the Institute of Biotechnology of the Universidad Nacional de Colombia, and in the Toxins Analysis Laboratory of the Institute of Biotechnology of the Universidad Nacional Autónoma de México. The test in mice were analyzed with albinos Mus musculus CD1 weighing between 18-20 g intraperitoneally (IP). Venom proteins and antivenoms were quantified by Absorbance 280 nm, Bradford method and Bicinchoninic acid (BCA). Polyacrylamide gel electrophoresis was performed in the presence of sodium duodecyl sulfate (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC) to analyze the protein profile. The mean lethal dose (LD50) of the venom and the mean effective dose (ED50) of the antivenoms were calculate. The immunochemical specificity and affinity of the antivenoms for the venom were evaluated with affinity chromatography, Western Blot, enzyme-linked immunosorbent assay (ELISA), and SDS-PAGE of the fractions coupled to each antivenoms. Results. The B. rhombeatus´s LD50 was 6,6 mg/kg, of B. asper was 6,4 mg/kg and of B. atrox was 6,7 mg/kg. The INS´s ED50 was 119,7 g/3LD50 and AVP-T´s ED50 was 270 g/3LD50 with B rhombeatus venom. Compared with B. asper venom, the INS´s ED50 was 62,74 g/3LD50 and AVP-T´s ED50 was 143,8 g/3LD50, and with B atrox venom the INS´s ED50 was 75,15 g/3LD50 and AVP-T´s ED50 was 190,2 g/3LD50. The electrophoretic profile and the chromatogram fractions of the venom was correlated, suggesting phospholipases A2 (PLA2), metalloproteinases (svMP) type I, II, III and serinoproteases (SP) in B. rhombeatus venom. Similarities of high, medium and low molecular weight protein were found between the profile of the three venoms with bands between 50-70 KDa corresponding to svMP III, band around 20-25 KDa of svMP I and between 11-15 KDa of PLA2. Both antivenoms showed immunochemical recognition towards PLA2 and svMP but at different intensity. INS showed recognition to svMP III fractions of B. atrox venom, same as towards svMP I and III of B. rhombeatus venom and svMP I, II and III fractions of the B. asper venom. Unlike INS, AVP-T showed immunochemical recognition to SP of B. rhombeatus venom and almost all the svMP fractions of B. asper venom, with higher intensity to svMP III and PLA2 of B. atrox venom. The quantification of antibodies coupled was for INS to B. rhombeatus was 94,2% and to B. asper venom was 92,7%; while AVP-T had 90,4% coupling to B rhombeatus and 96,6% to B. asper venom. These results are consistent with the ED50, because both antivenoms were effective to neutralize the lethality of the B. rhombeatus venom, however, considering neither was developed with this venom, thus inferring cross-reactivity towards PLA2 and svMP. All the above, were corroborated with the recognition level of the ELISA test, which it is evident that more INS antivenom was required to reach half of the maximum response with the B. rhombeatus venom compared to AVP-T. Conclusion. The results obtained suggest that both antivenoms may be a therapeutic option to the treatment of snakebite caused by B. rhombeatus, however, beyond that there is the fact is necessary to improve the specificity of commercial antivenoms, so that they are more related to the venom´s fractions with a significant effect on the pathophysiology of the patient, which would allow ophidiotoxicosis to be more easily neutralized without requiring a large amount of antivenoms and reducing secondary reactions to excess heterologous plasma immunoglobulins.