Immunoenzymatic quantitation of antibodies to Bothrops asper myotoxins after polyvalent antivenom administration in mice.

1. Two quantitative enzyme-immunoassays (EIA) for Bothrops asper myotoxin and anti-myotoxin antibodies, respectively, were utilized to study their in vivo distribution in mice (Swiss, 18 to 20 g). 2. After polyvalent antivenom (0.4 ml) administration by the iv route, there was an immediate peak in plasma anti-myotoxin antibodies which declined rapidly during the first hour, and then decreased more gradually. Anti-myotoxin antibodies were detected in muscular tissue (gastrocnemius) following iv injection of antivenom. After im injection of antivenom (0.4 ml), a slow and steady increase in plasma anti-myotoxin levels was observed, with a peak at 24 h. 3. Mice that received antivenom (0.4 ml) by the iv or im route 15 min after im injection of B. asper venom (100 micrograms) had lower levels of plasma anti-myotoxin antibodies than controls injected with antivenom only, suggesting that at least a fraction of the antibodies combines with myotoxins in vivo. 4. Myotoxin was not detected in plasma at any time after venom injection by the im (100 micrograms) or ip (40 micrograms) route. Following iv injection of 50 micrograms of purified myotoxin II, all plasma samples were also negative, at a detection limit of 10 ng/ml. 5. It was demonstrated that myotoxin II binds to mouse erythrocytes in vitro, a fact that could partially explain its rapid in vivo disappearance from plasma. 6. The present results on the distribution of anti-myotoxin antibodies in vivo are in agreement with previous experimental studies reporting the poor neutralization of myotoxicity induced by B. asper venom when antivenom is injected im, in comparison to i.v. injection.

Immunoenzymatic quantitation of antibodies to Bothrops asper myotoxins after polyvalent antivenom administration in mice

Two quantitative enzyme-ummunoassays (EIA) for Bothrops asper myotoxin and anti-myotoxin antibodies, respectively, were utilized to study their in vivo distribution in mice (Swiss, 18 to 20 g). After polyvalent antivenom (0.4 ml) administration by the iv route, there was an immediated peak in plasma anti-myotoxin antibodies which declined rapidly during the first hour, and then decreased more gradually. Anti-myotoxin antibodies were detected in muscular tissue (gastrocnemius) following iv injection of antivenom. After im injection of antivenom (0.4 ml), a slow and steady increase in plasma anmti-myotoxin levels was observed, with a peak at 24 h. Mice that received antivenom (0.4 ml) by the iv or im route 15 min after im injection of B. asper venom (100 ug) had lower levels of plasma anti-myotoxin antibodies than controls injected with antivenom only, suggesting that at least a fraction of the antibodies combines with myotoxins in vivo. Myotoxin was not detected in plasma at any time after venom injection by the im (100 ug) or ip (40 ug) route. Following iv injection of 50 ug of purified myotoxin II, all plasma samples were also negative, at a detection limit of 10 ng/ml. It was demonstrated that myotoxin II binds to mouse erythrocytes in vitro, a fact that could partially explain its rapid in vivo disappearance from plasma. The present results on the distribution of anti-myotoxin antibodies in vivo are in agreement with previous experimental studies reporting the poor neutralization of myotoxicity induced by B. asper venom when antivenom is injected im, in comparison to iv injection

Equine antibodies to Bothrops asper myotoxin II: isolation from polyvalent antivenom and neutralizing ability.

Equine antibodies to B. asper myotoxin II were isolated from polyvalent antivenom by affinity chromatography. Purified antibodies were among the most acidic serum immunoglobulins, migrating between the beta- and alpha 2-globulin regions by zone electrophoresis. This might be related to the fact that myotoxin II is a very basic antigen. At an antibody/toxin molar ratio of two or higher, myotoxic effect of myotoxin II was neutralized completely. Antibodies also neutralized myotoxic activity of crude venom in mice, reducing the effect by about 75% when a proportion of 16 mg antibodies/mg venom was tested. Despite the lack of phospholipase A2 activity of myotoxin II, antibodies were able to neutralize this enzymatic activity of myotoxin I, a previously described isoform. This finding is in agreement with the notion that myotoxin II is a phospholipase A2-analog devoid of this enzyme activity.

Isolation of basic myotoxins from Bothrops moojeni and Bothrops atrox snake venoms.

Three myotoxins, one from the venom of Bothrops atrox and two from the venom of B. moojeni, were isolated by ion-exchange chromatography on CM-Sephadex C-25. The three toxins are basic proteins with an estimated mol. wt of about 13,500, and similar amino acid compositions. When injected into the gastrocnemius muscle of mice, the three toxins induce drastic myonecrosis of rapid onset, as judged by histological observation and quantitation of plasma creatine kinase levels. B. atrox myotoxin also has phosphlipase A2 and anticoagulant activities, whereas B. moojeni myotoxins I and II lack these effects. The three toxins are antigenically similar to each other, and to previously isolated myotoxins I and II from the venom of B. asper, when tested by gel immunodiffusion against rabbit antiserum to B. asper myotoxin I. Two monoclonal antibodies against B. asper myotoxins were tested against the newly purified proteins. MAb-3 recognizes all of them, whereas MAb-4 recognizes only B. atrox myotoxin, by enzyme-immunoassay. B. atrox and B. moojeni myotoxins can be tentatively classified within a group of myotoxins having phospholipase A2 structure present in Bothrops venoms.

South American snake venom proteins antigenically related to Bothrops asper myotoxins.

1. The presence of proteins antigenically related to Bothrops asper myotoxins in various snake venoms, mainly from South America, was investigated by using polyclonal and monoclonal antibodies. 2. Myotoxin-like components were detected in ten Bothrops venoms from South America, and in the venoms of Crotalus atrox (North America), Trimeresurus flavoviridis (Japan), and Micrurus alleni (Costa Rica). 3. Cross-reactive components detected in several Bothrops venoms show a common subunit of 15-16 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, although significant charge variations are evident by immunoelectrophoresis. 4. It is concluded that proteins antigenically related to B. asper myotoxins are relatively common in the genus Bothrops and, in the light of findings discussed, are likely to possess myotoxic activity.

South American snake venom proteins antigenically related to bothrops asper myotoxins

1. the presence of proteins antigenically related to Bothrops asper myotoxins in various snake venoms, mainly from South America, was investigated by using poluclonal and monoclonal antibodies. 2. Myotoxin-like components were detected in the bothrops venoms from South america, and in the venoms of Crotalus atrox (North america), Trimerusurus flavoviridis (Japan), and Micrurus alleni (Costa Rica). 3. Cross-reactive components detected in several Bothrops venoms show a common subunit of 15-16 LDa by sodium dodcyl sulphate-polyacrylamide gel electrophoresis, although significant charge variations are evident by immunoelectrophoresis. 4. It is concluded that proteins antigenically related to B. asper nyotoxins are relatively common in the genus Bothrops and, in the light of findings discussed, are likely to posses myotoxic activity