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BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K(d)), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K(d)s of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation., ((c) 2010 Elsevier Ltd. All rights reserved.)

Bothropic antivenom and its IgG(T) fraction, administered 4 h after experimental envenoming by Bothrops jararaca in Swiss mice, were compared for their abilities to restore fibrinogen 24 or 48 h after treatment. IgG(T) was able to normalise fibrinogen levels as efficiently as conventional antivenom. As IgG(T) also neutralises most anti-toxic activities of Bothrops venom, our results suggest that IgG(T) could be a better alternative treatment for envenoming due to the reduced amount of extraneous proteins, which may facilitate the induction of early adverse reactions.

Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 000-000. This work compared the specificity, ELISA titers and IgG subclass content of the polyvalent antivenom (anti-Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys) of Instituto Clodomiro Picado (Costa Rica) and the bothropic antivenom (anti-Bothrops jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus) of Instituto Butantan (Brazil). The role of IgG(T) and IgGa subclasses in neutralization of some venom toxic activities and the cross neutralization of the antivenoms against B. jararaca and B. asper venoms were also evaluated. Both antivenoms were able to recognize B. asper and B. jararaca venoms by immunoblotting and presented similar antibody titers when assayed by ELISA. IgG(T) was highest, followed by IgGa, IgGb and IgGc. IgGa and IgG(T) isotypes isolated from both antivenoms by affinity chromatography were tested for neutralization of lethal, hemorrhagic, coagulant and phospholipase A2 activities of the homologous venoms. In both antivenoms, IgG(T) was the major isotype responsible for neutralization of all the tested activities, followed by IgGa. These results suggest that Instituto Butantan and Instituto Clodomiro Picado antivenoms have the same IgG profile and their neutralizing ability is due mostly to the IgG(T) isotype. Also, they neutralize lethality in mice induced by homologous and heterologous venoms, the bothropic antivenom of Instituto Butantan being more effective.

IgG(T) and IgGa isotypes were isolated from horse hyperimmune anti-bothropic and anti-crotalic sera using a combination of two affinity chromatographic processes. IgG(T) and IgGa isotypes were isolated from these sera by chromatography on protein A-Sepharose followed by separation of the two isotypes by chromatography on a column of anti-IgG(T)-Sepharose. LO-HoGT-1, a rat anti-horse IgG(T) monoclonal antibody, was used. A comparative study of the efficiency of these isotypes in neutralizing the main toxic activities of the homologous venoms was carried out. It was found that IgG(T) was about three-fold and seven-fold more protective than IgGa for neutralization of the lethal activity of B. jararaca and C. d. terrificus venoms, respectively. IgG(T) was also more effective than IgGa for the neutralization of the haemorrhagic activity induced by B. jararaca venom, while both isotypes neutralized equally well the blood incoagulability induced by this venom. The results suggest that IgG(T) is the most protective isotype present in both anti-bothropic and anti-crotalic sera, followed by IgGa. Owing to their very low concentration in the serum, other IgG isotypes are not likely to be important in neutralizing the venoms' toxic activities.

1. Bothrops jararaca venom was detected by ELISA at different times in the skin, muscle, blood, liver, lung, heart, kidney and spleen of mice injected with venom i.m. or i.d. 2. The results showed that even 10 min after i.m. injection the venom is detected mostly in skin rather than in the muscle of the venom injection site. A small amount of venom was detected in the kidney up to 12 h after im venom injection, and none was detected in tissues of lung, heart, liver or spleen. 3. However, in mice injected i.d., the venom could be detected in the skin up to 24 h after injection. Local necrosis and haemorrhage could be neutralized by antivenom injected by the i.d. or i.v. routes only if the antivenom was given a short time after venom injection, even when antivenom is administered in high concentration. 4. In contrast, experiments performed in mice receiving venom i.d. and treated by i.d. or i.v. routes with antivenom injected at different times after envenoming showed that the effect of venom on blood coagulation could be counteracted by antivenom administered by either route up to 2 h after venom injection. 5. We suggest that a feasible amount of antivenom administered i.d. could be given as a first aid measure after a snake bite accident. However, further experimental studies using the i.d. route for antivenom administration are essential to confirm this possibility.