Antibody from mice immunized with DNA encoding the carboxyl-disintegrin and cysteine-rich domain (JD9) of the haemorrhagic metalloprotease, Jararhagin, inhibits the main lethal component of viper venom.

Envenoming by the Brazilian pit viper, Bothrops jararaca, induces extensive local and systemic haemorrhage in humans. The severe and occasionally lethal outcome of envenoming is prevented only by administration of antivenom which is conventionally prepared by hyperimmunization of large animals with an individual venom or a range of venoms. Since snake venoms typically consist of numerous molecules, only some of which are toxic, antivenoms are antigenically crude preparations whose therapeutic value would theoretically be enhanced by restricting antibody specificity to toxic venom molecules. We report here that high-titre IgG antibody from mice immunized by the GeneGun with DNA encoding the carboxy-terminal JD9 domain of Jararhagin, a haemorrhage-inducing metalloprotease in B. jararaca venom, extensively neutralized the main lethal component of B. jararaca venom. This is to our knowledge the first study to apply DNA-based methods to preparation of antivenom; it represents a novel approach with greater immunological specificity and fewer hazards than conventional systems of antivenom production.

California ground squirrel (Spermophilus beecheyi) blood sera inhibits crotalid venom proteolytic activity.

Some California ground squirrels (Spermophilus beecheyi) show limited necrosis following envenomation by northern Pacific rattlesnakes (Crotalus viridis oreganus). This study demonstrates that S. beecheyi blood sera inhibits venom proteases. Sera from rattlesnake-abundant habitats inhibited C. v. oreganus venom more effectively than venom from two allopatric rattlesnake species, C. v. viridis and C. atrox, suggesting evolutionary specialization. The pattern of inhibition among squirrel populations corresponds best with history of rattlesnake predation, in contrast to current rattlesnake density.

Determination of the neutralizing potency of horse antivenom against bothropic and crotalic venoms by indirect enzyme immunoassay.

The use of ELISA to determine antisnake venom potency of horse immune sera should provide benefits of costs and reproducibility compared to in vivo assays. In the present investigation we evaluated the correlation between ELISA antibody levels and in vivo neutralization assays. For the indirect ELISA method, 0.016 micrograms/well of Bothrops jararaca or Crotalus durissus terrificus venom were used to coat the plates and 100 microliters/well of each sample of antibothropic or anticrotalic venom sera were used at 1:10,000 dilution. Sheep anti-horse IgG conjugated to peroxidase was added and the substrate H2O2/o-phenylenediamine produced the color that was read at 492 nm. A correlation coefficient of r = 0.97 was found for anticrotalic venom antibodies and no significant correlation was observed for antibothropic venom sera using 16 serum samples from immunized horses. However, when three antibothropic venom sera showing high in vivo neutralization potency and low absorbance in ELISA or high absorbance values and low in vivo protection were not included in the correlation analysis the coefficient value was r = 0.88. The correlation coefficient did not improve for all 16 antibothropic sera when a partially purified Bothrops jararaca venom fraction was used to coat the ELISA plates. The results indicate that ELISA could be used to determine the neutralizing potency of anticrotalic venom sera. For the antibothropic venom sera further studies are needed.

Determination of the neutralizing potency of horse antivenom against bothropic and crotalic venoms by indirect enzyme immunoassay

The use of ELISA to determine antisnake venom potency of horse immune sera should provide benefits of cost and reproducibility compared to in vivo assays. In the present investigation we evaluated the correlation between ELISA antibody levels and in vivo neutralization assays. For the indirect ELISA method, 0.016 µg/well of Bothrops jararaca or Crotalus durissus terrificus venom were used to coat the plates and 100 µl/well of each sample of antibothropic or anticrotalic venom sera were used at 1:10,000 dilution. Sheep anti-horse IgG conjugated to peroxidase was added and the substrate H202/o-phenylenediamine produced the color that was read at 492 nm. A correlation coefficient of r=0.97 was found for anticrotalic venom antibodies and no significant correlation was observed for antibothropic venom sera using 16 serum samples from immunized horses. However, when three antibothropic venom sera showing high in vivo neutralization...

An indirect haemolytic assay for assessing antivenoms.

Dilutions of antivenom, venom, human erythrocytes and a phosphatidylcholine suspension, were incubated for 30 min at 37 degrees C. After centrifugation, the liberated haemoglobin was measured spectrophotometrically. The assay was used to assess an ovine antivenom against the venom from the South American rattlesnake, Crotalus durissus terrificus, and an equine Wyeth antivenin (Crotalidae, polyvalent). The ovine antivenom was more than five times as effective as the equine product. It also neutralized venoms from the Western diamondback rattlesnake, Crotalus atrox, and the fer-de-lance, Bothrops atrox. However, antivenoms raised against venoms from other Crotalus and Bothrops species provided little protection against the haemolytic activity of C. d. terrificus venom.

Neutralización de las actividades tóxicas y enzimáticas de cuatro venenos de serpientes de Guatemala y Honduras por el antiveneno polivalente producido en Costa Rica / Neutralization of toxic and enzyme activities of 4 venoms from snakes of Guatemala and Honduras by the polyvalent antivenin produced in Costa Rica

Se estudió la capacidad del antiveneno polivalente producido en Costa Rica para neutralizar las actividades letal, hemorrágica, edematizante, proteolítica, hemolítica, hialuronidasa y fibrinolítica de los venenos de Bothrops asper y Bothrops nummifer de Honduras y de Agkistrodon bilineatus y de Crotalus durissus durissus de Guatemala. La capacidad neutralizante del suero se expresó como DE50 (Dosis efectiva 50%), definida como la razón antiveneno/veneno en la que la actividad del veneno es reducida en un 50%. El antiveneno fue altamente eficaz en la neutralización de las actividades letal, hemorrágica, hemolítica, hialuronidasa y caseinolítica de los venenos de B. asper, B. no de B. nummifer, el efecto fibrinolítico fue neutralizado sólo parcialomente, en tanto que estudiaron los venenos de B. asper y C. d. durissus. El antiveneno neutralizó adecuadamente el veneno de A. bilineatus con excepción del efecto hemolítico, cuya neutralización fue sólo parcial. Sin embargo, en términos cuantitativos, se requirió un volumen mayor de antiveneno para neutralizar algunas actividades del veneno de A. bilinneatus. En relación al efecto edematizante, el antiveneno neutralizó eficazmente los venenos de B. asper y A. bilineatus, en tanto que el veneno de B. nummifer fue neutralizado sólo parcialmente, y el de C. d. durissu no fue neutralizado del todo. Experimentos inmunoquímicos mostraron una gran similitud entre los venenos de B. asper, B. nummifer y C. d. durissus colectados en Honduras y Guatemala cuando se compararon con los de las mismas especies colectadas en Costa Rica. No bostante, hay divergencias inmunoquímicas entre venenos de diferentes especies. Se concluye que, con la excepción del efecto edematizante inducido por los venenos de B. nummifer y C. d. durissus, el antiveneno polivalente producido en costa Rica es efectivo en la neutralización de los venenos de estas cuatro especies de serpientes