Neutralización de la actividad letal del veneno de serpiente Bothrops atrox por suero hiperinmune de llama (Lama glama) / Neutralization of the lethal activity from Bothrops atrox venom by hyperimune llama serum (Lama glama)

RESUMEN Objetivos: Evaluar la capacidad del suero hiperinmune de llama (Lama glama) para neutralizar la letalidad del veneno de la serpiente Bothrops atrox en ratones de laboratorio. Materiales y métodos: Se calculó la dosis letal media (DL50) de un pool de venenos de serpientes de Bothrops atrox de Perú, y se midieron los títulos de anticuerpos por ensayo ELISA; así como la potencia de neutralización del suero inmune por el cálculo de la dosis efectiva media (DE50) durante el periodo de inmunización. Resultados: La DL50 del veneno fue de 3,96 µg/g, similar a otros trabajos realizados en Bothrops atrox en Perú. Los títulos de anticuerpos contra el veneno se incrementan rápidamente en la llama mostrando una rápida respuesta inmune; sin embargo, la capacidad de neutralización se incrementa más lentamente y requiere de varias dosis y refuerzos de las inmunizaciones alcanzado una DE50 de 3,30 µL/g ratón y una potencia de neutralización 3,6 mg/mL después de 15 inmunizaciones. Conclusiones: El suero hiperinmune de llama es capaz de neutralizar la letalidad del veneno de la serpiente Bothrops atrox de Perú en ratones de laboratorio.

ABSTRACT Objectives: To evaluate the capacity of the hyperimmune llama serum (Lama glama) to neutralize the lethal activity of Bothrops atrox venom in laboratory mice. Materials and methods: Mean lethal dose (LD50) was calculated from a Bothrops atrox venom sample pool from Peru. The antibody titers were measured by ELISA assay; and the immune serum neutralization potency was measured by calculating the mean effective dose (ED50) during the immunization period. Results: The venom's LD50 was 3.96 μg/g; similar to what was found in other studies about Bothrops atrox carried out in Peru. The titers of antibodies against the venom increased rapidly in the llama, demonstrating a fast immune response; however, the neutralization capacity increased slowly and required several doses and immunization reinforcements, obtaining a ED50 of 3.30 μL/g mouse and a neutralization potency of 3.6 mg/mL after 15 immunizations. Conclusions: The hyperimmune llama serum is able to neutralize the lethality of the Bothrops atrox venom from Peru in laboratory mice.

Preclinical efficacy against toxic activities of medically relevant Bothrops sp. (Serpentes: Viperidae) snake venoms by a polyspecific antivenom produced in Mexico / Eficacia de un antiveneno poliespecífico producido en México para neutralizar, a nivel preclínico, las actividades tóxicas de venenos de Bothrops sp (Serpentes: Viperidae) de importancia médica

Abstract:The assessment of the preclinical neutralizing ability of antivenoms in Latin America is necessary to determine their scope of efficacy. This study was aimed at analyzing the neutralizing efficacy of a polyspecific bothropic-crotalic antivenom manufactured by BIRMEX in Mexico against lethal, hemorrhagic, defibrinogenating and in vitro coagulant activities of the venoms of Bothrops jararaca (Brazil), B. atrox (Perú and Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia), and B. asper (Costa Rica). Standard laboratory tests to determine these activities were used. In agreement with previous studies with bothropic antivenoms in Latin America, a pattern of cross-neutralization of heterologous venoms was observed. However, the antivenom had low neutralizing potency against defibrinogenating effect of the venoms of B. atrox (Colombia) and B. asper (Costa Rica), and failed to neutralize the in vitro coagulant activity of the venom of B. asper (Costa Rica) at the highest antivenom/venom ratio tested. It is concluded that, with the exception of coagulant and defibrinogenating activities of B. asper (Costa Rica) venom, this antivenom neutralizes toxic effects of various Bothrops sp venoms. Future studies are necessary to assess the efficacy of this antivenom against other viperid venoms. Rev. Biol. Trop. 65 (1): 345-350. Epub 2017 March 01.

ResumenEs necesario estudiar a nivel preclínico la capacidad neutralizante de los antivenenos producidos en América Latina, para conocer su espectro de cobertura. En este estudio se analizó la eficacia preclínica de un antiveneno poliespecífico botrópico-crotálico producido por BIRMEX, en México, para neutralizar los efectos letal, hemorrágico, desfibrinogenante y coagulante in vitro de los venenos de Bothrops jararaca (Brasil), B. atrox (Perú y Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia) y B. asper (Costa Rica). Se emplearon metodologías de laboratorio estándar en los análisis. En consonancia con estudios anteriores con diversos antivenenos botrópicos en América Latina, se observó un amplio patrón de neutralización de estos venenos heterólogos en la mayoría de los efectos estudiados. Sin embargo, el antiveneno mostró una baja capacidad neutralizante contra el efecto desfibrinogenante de los venenos de B. atrox (Colombia) y B. asper (Costa Rica) y no neutralizó la actividad coagulante in vitro del veneno de B. asper (Costa Rica) a la máxima razón antiveneno/ veneno empleada.

Eficacia experimental de anticuerpos IgY producidos en huevos, contra el veneno de la serpiente peruana Bothrops atrox / Experimental efficacy of IgY antibodies produced in eggs against the venom of the Peruvian snake Bothrops atrox

Objetivos. Desarrollar un protocolo de inmunización para producir inmunoglobulinas IgY de origen aviar contra el veneno de la serpiente peruana Bothrops atrox y evaluar la capacidad neutralizante. Materiales y métodos. Se inmunizaron seis gallinas de postura de la raza hy line brown con 500 μg/dosis de veneno de B. atrox en un periodo de dos meses. Cada semana, los huevos fueron colectados para el aislamiento de inmunoglobulinas IgY a partir de la yema, usando dos pasos consecutivos con αcido caprνlico y sulfato de amonio. La detecciσn de anticuerpos se realizσ por inmunodifusiσn doble mientras que el tνtulo y reactividad cruzada se determinaron por las técnicas de ELISA y Western blot. El cálculo de DL50 y de la DE50 del antiveneno IgY producido se realizó utilizando el método de Probits. Resultados. La masa de anticuerpos aislados fue de 8,5 ± 1,35 mg de IgY/mL de yema. Asimismo, la DE50 del antiveneno aviar fue calculada en 575 μL de antiveneno/mg de veneno. Adicionalmente, los ensayos de reactividad cruzada mostraron que el veneno de B. atrox comparte mas epνtopes comunes con el veneno de B. brazili (47%) que con otros veneno del mismo género, en tanto que los venenos de Lachesis muta (19%) y Crotalus durissus (12%) mostraron una baja reactividad cruzada. Conclusiones. Se ha obtenido IgY purificada contra el veneno de B. atrox con capacidad neutralizante y se ha demostrado su utilidad como herramienta inmunoanalítica para evaluar la reactividad cruzada con venenos de otras especies.

Objectives. To develop an immunization protocol in order to produce avian IgY immunoglobulins against Bothrops atrox Peruvian snake venom and to evaluate its neutralizing capacity. Materials and methods. Six Hy Line Brown hens were immunized each two weeks using 500μg/doses of B. atrox venom in a period of two months. Each week, eggs were collected for IgY isolation from yolk using two consecutive steps with caprilic acid and ammonium sulfate. Detection of IgY anti-B. atrox were performed by double immunodiffusion, whereas title and cross-reactivity were analyzed using ELISA and Western Blot technics, respectively. Furthermore, letal dose (DL50) and Medium Effective Dose (DE50) were obtained by Probit analysis. Results. As a result of this protocol, chicken IgY’s were obtained in a concentration of 8,5 ± 1,35 mg/yolk mL. DE50 from avian antivenom was 575 μL/venom mg. Cross-reactivity studies showed Bothrops atrox venom share more commom epitopes with Bothrops brazili (47%) than others Bothrops venoms showing Lachesis muta (19%) and Crotalus durissus (12%) venoms a low crossing reactivity, instead. Conclusions. Using this procedure, we could purify chicken IgY with a neutralizant capacity of B. atrox venom which is comparable to the antivenom of equine origin and demonstrate its capacity as a immunoanalitical tool to evaluate the cross reactivity with others peruvian snakes.

Cross-neutralization of the coagulant activity of Crotalus durissus terrificus venom from the northeast of Argentina by bivalent bothropic antivenom

Cross-neutralization of Crotalus durissus terrificus venom coagulant activity was tested using bivalent horse antivenom against Bothrops alternatus and Bothrops diporus venoms. Our in vitro and in vivo experiments showed that bothropic antivenom neutralizes the thrombin-like activity of crotalic snake venom and this cross-reaction was demonstrated by immunoassays either with whole venom or a purified thrombin-like enzyme. These results suggest common antigenic properties and, consequently, similar molecular structure among venom thrombin-like enzymes. Besides, they provide information that could be further used in the development of new antivenom formulations.

Humoral immune response of patients bitten by the snake Bothrops erythromelas / Resposta imune humoral em pacientes picados pela serpente Bothrops erythromelas

INTRODUCTION: Snake envenomings are a health problem in rural areas of tropical and subtropical countries, but little is known regarding the immune response presented by bitten individuals. The IgM production of patients bitten by Bothrops erythromelas snake was analyzed to identify the effectiveness of treatment in this type of envenomation. METHODS: Bothrops erythromelas venom was submitted to electrophoresis and transferred to a nitrocellulose sheet, following incubation with patients' sera. RESULTS: A 38 KDa protein was detected before and 24 h after therapy. CONCLUSIONS: The result suggests that this protein could be used as a marker for individuals envenomed by Bothrops. erythromelas.

INTRODUÇÃO: Envenenamentos ofídicos consistem problema de saúde pública em áreas rurais de países tropicais e subtropicais, mas pouco sabe-se sobre a resposta imune apresentada pelos indivíduos picados, por isso a avaliação da produção de IgM por pacientes picados por Bothrops erythromelas identificando a eficácia do tratamento nesse tipo de envenenamento. MÉTODOS: O veneno de Bothrops erythromelas foi submetido a eletroforese e transferido para nitrocelulose, seguindo incubação com soro de pacientes. RESULTADOS: Foi observada proteína de 38KDa antes e 24 horas após o tratamento. CONCLUSÕES: Os resultados sugerem que essa proteína poderia ser utilizada como marcador para indivíduos envenenados pela serpente Bothrops erythromelas.

Hiperinmunización de ovinos contra veneno de Bothrops asper del estado Zulia Venezuela: estudio preliminar / Hyperimmunization of ovines against Bothrops asper venom from the Zulia state Venezuela: preliminary study

Un grupo de cuatro ovinos sanos se inmunizó con veneno de Bothrops asper, para estudiar el desarrollo de la respuesta inmune, inducida por la aplicación de un esquema de hiperinmunización. Se tomaron muestras de sangre cada siete días en nueve oportunidades, con el suero obtenido se realizaron pruebas de neutralización en ratones. Se determinó la DE 50, la cual se expresó en µg de veneno neutralizado por ml de suero. Simultáneamente se observaron las condiciones generales de los ovinos durante el esquema de hiperinmunización, no presentándose alteraciones generales ni locales como consecuencia de la inoculación del veneno. En los resultados se observaron variaciones individuales en la magnitud de la elevación del título de anticuerpos y rasgos comunes en el comportamiento de las curvas desarrolladas. El título promedio óptimo (705,5 µg/ml), fue obtenido el día 21 posterior a la primera inoculación. Para ese momento, el ovino con título más alto fue de 840µg/ml y el más bajo, de 593 µg/ml. El promedio más bajo (308 µg/ml) fue observado el día 56. Se recomienda que los animales que serán utilizados en la producción comercial de antiveneno sean evaluados individualmente y seleccionados con base a una respuesta inmune satisfactoria.

A group of four (4) healthy male sheep were immunized with Bothrops asper venom, in order to study the development of the immune response induced by the application of a hiperimmunization protocol. Blood samples were taken from the sheep every seven days in nine different opportunities. With the resultant serum, neutralization tests were done in mice. ED50 was calculated and expressed in µg of neutralized venom per ml of serum, at the same time, general health conditions of sheep were observed during the hiperimmunization protocol. No general or local alterations of health were present as consequence of venom inoculations. Individual variations in the magnitude of the increase of antibody titles were observed in the obtained results. None the less common features in the antibody title curves were also recorded. The mean optimal average (705,5 µg/ml) was obtained on day 21 after the first challenge. At that time, the individual sheep with the highest value was 840 µg/ml and the lowest was 593 µg/ml; the lowest average of all samples (308 µg/ml ) was observed on day 56. It is recommend that animals to be used in the commercial venom antiserum production need to be individually screened and selected based on a satisfactory immune response.

Características biológicas e inmunológicas del veneno de Bothrops cotiara (Serpentes: Viperidae) / Biological and immunological characteristics of the poison of Bothrops cotiara (Serpentes: Viperidae)

Bothrops cotiara is a venomous snake sporadically found in the province of Misiones in Argentina, South of Brazil and Paraguay. Data on the clinics of the envenomation produced by its bite and on its venom are scarce. There is no information on the neutralizing capacity of the antivenoms available. In this study, the lethal potency, hemorrhagic, necrotizing, coagulant and thrombin-like, defibrinogenating, indirect hemolytic and fibrinolytic activities of the venom of B. cotiara specimens from the province of Misiones were determined. The toxic activities were within the range of those described for the other Bothrops species from Argentina, and the electrophoretic and chromatographic studies showed similarities with those described for the other bothropic venoms. The immunochemical reactivity of six South American anti Viper antivenoms (ELISA) have a strong reactivity with all the antivenoms studied. The neutralizing capacity of three of these therapeutic antivenoms against the lethal potency and hemorrhagic, necrotizing, coagulant, thrombin-like and hemolytic activities showed a very close neutralizing capacity. Our data strongly suggest that the antivenoms for therapeutic use available in this area of South America are useful to neutralize the toxic and enzymatic activities of the venom of this uncommon specie of Bothrops.

Bothrops cotiara es una serpiente que se encuentra en la provincia de Misiones (Argentina), el Sur de Brasil y Paraguay. La información sobre las características clínicas de los accidentes por esta serpiente es muy escasa y existen pocos datos sobre su veneno y la capacidad neutralizante de las actividades tóxicas del mismo por antivenenos terapéuticos. En este trabajo se estudiaron características bioquímicas, actividades tóxicas y la reactividad inmunoquímica del veneno de B. cotiara. Seis antivenenos anti Viperinos Sudamericanos fueron estudiados frente a este veneno por el método ELISA y se probó la capacidad neutralizante de tres de estos frente a las actividades hemorrágica, necrotizante, procoagulante, trombina-símil, hemolítica indirecta y la potencia letal de veneno de ejemplares de B. cotiara de la provincia de Misiones. Los patrones cromatográficos y electroforéticos mostraron características similares a los de otros venenos de Bothrops. Las actividades tóxicas estuvieron dentro de los ámbitos descritos para los venenos botrópicos. Los seis antivenenos mostraron gran reactividad inmunoquímica por ELISA y las potencias neutralizantes de los tres estudiados fueron muy próximas para las actividades letal, hemorrágica, necrotizante, hemolítica indirecta, coagulante y trombina-símil. Los resultados de los estudios de neutralización indicarían que ante la mordedura de esta poco común especie de Bothrops, pueden usarse los diferentes tipos de antivenenos botrópicos o botrópico-crotálicos para uso terapéutico disponibles en esa región.

Rabbit IgG antibodies against Phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms

El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 µg.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes

Aspectos toxinológicos e inmunoquímicos del veneno del escorpión Tityus pachyurus Pocock de Colombia: capacidad neutralizante de antivenenos producidos en Latinoamérica / Toxicological and immunological aspects of scorpion venom ( Tytius pachyurus): neutralizing capacity of antivenoms produced in Latin America

Este trabajo se realizó con el objetivo de determinar la toxicidad y las características inmunoquímicas del veneno del escorpión Tityus pachyurus y su neutralización por tres antivenenos antiescorpión producidos en Latinoamérica: Alacramyn® del Instituto Bioclón de México; el suero antiescorpiónico del Instituto Butantán de São Paulo, Brasil, y el suero antiescorpiónico del Centro de Biotecnología de la Universidad Central de Venezuela, Caracas, Venezuela. Este escorpión produjo 0,68±0,20 mg (promedio ± DE) de veneno por estimulación manual, el cual presentó una dosis letal 50% (DL50) en ratones de 4,8 myg/kg (91,3 myg/ratón de 18-20 g). Los signos de envenenamiento predominantes en los ratones fueron: sialorrea, dificultad respiratoria, sudoración generalizada, ataxia, alteraciones del comportamiento (excitabilidad, somnolencia) e hiperglicemia 3 y 24 horas después de la inyección 0,5 DL50 del veneno por vía subcutánea. Para los antivenenos de México y Brasil, la dosis efectiva 50% neutralizante del efecto letal fue de 330 y 292 myg de veneno por ml de antiveneno, respectivamente. El antiveneno de Venezuela no neutralizó este efecto. Por electroforesis (SDS-PAGE) se demostró que el veneno contiene proteínas desde menos de 14 kd hasta 97 kd. Los Western blot indicaron reactividad inmunológica de los tres antivenenos con los diversos componentes del veneno, incluso las proteínas de baja masa molecular (<14 kd). Los resultados permiten concluir que el veneno de T. pachyurus es neutralizado eficientemente por los antivenenos contra picaduras de escorpiones producidos en México y Brasil.

The toxicity and immunochemical properties of Tityus pachyurus Pocock scorpion venom was characterized, as well as the neutralization capacity against it by three anti-scorpion antivenoms (Alacramyn®, Instituto Bioclón, México; Suero antiescorpiónico, Instituto Butantán, Sao Paulo, Brasil; and Suero antiescorpiónico, Centro de Biotecnología, Universidad Central de Venezuela, Caracas, Venezuela). The venom yield, obtained by manual milking, 680±20 mug venom, a 50% lethal dose in mice was 4.8 mug/kg (90 mug for an 18-20 g mouse). The most common symptoms of venom poisoning in mice were sialorrhea, respiratory distress, profuse sweating, ataxia, behavior alterations (restlessness, somnolence) and hyperglycemia at 3 and 24 hours after subcutaneous venom injection (0.5 LD50). The neutralizing capacity of Bioclón (México City) and Butantán (Sao Paulo) antivenoms (for a 50% effective dose) was 330 and 292 mug venom/ml antivenom, respectively. The Biotecnología (Caracas) antivenom did not neutralize the lethal effect of venom. By electrophoresis (SDS-PAGE) was demonstrated that the venom contains proteins from less than 14 kd to 97 kd. The Western blots indicated immunological reactivity of the three antivenoms with most of venom components, including proteins of low molecular mass (<14 kd). The results allow to conclude that T. pachyurus venom is neutralized efficiently by anti-scorpion antivenoms produced in México and Brasil.

In vitro assay of biological and chemical toxins using antibodies against lethal toxin neutralizing factor

Lethal Toxin Neutralizing Factor (N-LTNF), MW 63.0 kDa, was isolated from opossum serum. After trypsin digestion, the active domain of N-LTNF was isolated and sequenced. The synthetic peptide consisting of ten amino acids was designated as LT-10. N-LTNF and LT-10 inhibited the lethality of animal, plant and bacteria toxins when tested on mice non-immunologically. The antibodies against N-LTNF and LT-10 only reacted immunologically with toxins and not with non-toxic substances. Anti-LTNF and anti-LT-10 reacted immunologically by ELISA test with toxins that were not detected by mouse test, such as cholera toxin and digoxin. Anti-LTNF and anti-LT-10 failed to react immunologically with non-toxic substances, such as nerve growth factor and collagen. Currently, mouse bioassay is in use for toxin detection and assay. Binding affinity of IgG from anti-LT-10 showed a linear relationship with mouse bioassay by ELISA detection limit to some toxins only. This may be due to the fact that anti-LTNF and anti-LT-10 detected the toxins that were not lethal to mouse. Thus, anti-LTNF and anti-LT-10 can be useful in assaying toxins as an alternative to mouse bioassay.

Estudo experimental da cinética do veneno e do antiveneno do escorpiäo Tityus serrulatus no soro e tecidos de camundongos / Experimental study of the kinetics of Tityus serrulatus scorpion venom in serum and tissues of mices

O presente trabalho descreve a farmacocinética (do envenenamento experimental) da peçonha do escorpiäo Tityus serrulatus, a farmacocinética do antiveneno escorpiônico e os efeitos da sua administraçäo sobre as concentraçöes do veneno no soro e em vários tecidos de camundongos da raça CF1, determinados através da técnica de ELISA descrita por Chávez-Olórtegui et al. (1994). Descreve também a distribuiçäo tecidual da peçonha do escorpiäo Tityus serrulatus determinada através de imunohistoquímica...

Standartization of an enzyme linked immunonosorbent assay (ELISA) for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus

Neste trabalho foram determinadas a sensibilidade e a especificidade da tecnica imunoenzimatica (ELISA) desenvolvida por CHAVEZ-OLORTEGUI et al. para detectar antigenos circulantes de veneno em pacientes picados por Tityus serrulatus. A media mais dois desvios padrao da observancia do soro de 100 pacientes controles foi utilizada como limite entre teste positivo e teste negativo ("cutoff"). A especificidade do ELISA foi igual a 97,0 por cento . A sensibilidade do metodo, quando incluidos pacientes classificados como casos leves, moderados e graves de escorpionismo, foi de 39,3 por cento e aumentou para 94,7 por cento quando considerados apenas os casos moderados e graves. Estes resultados mostram que o ELISA pode ser utilizado para deteccao de antigenos toxicos circulantes em pacientes com manifestacoes sistemicas de envenenamento escorpionico mas nao deve ser empregado no estudo de pacientes que apresentam apenas dor no local da picada (casos leves). O tempo necessario para a realizacao do ELISA e superior a 1 hora. Portanto, o teste tem sua utilizacao limitada para o diagnostico de envenenamento, mas pode construir um instrumento util para o estudo da cinetica de neutralizacao do veneno pelo antiveneno especifico.

Suppresion of the antivenom antibody response by serum therapy

1. The antivenom antibody response of mice injected with Bathrops jararaca venom and receiving specific serum therapy was studied under different experimental conditions. Balb/c mice (18-22g) injected with venom (1.75 mg/kg) presented the clinical symptoms observed in patients bitten by B. jararaca and a high and long-lasting antivenom antibody response. 2. Injection of 0.1 ml of horse antiserum to venom 15 min after venom administration abolished the symptoms induced by the venom and induced an almost completely suppressed production of mouse antivenom antibodies. The extent of suppression of the antivenom antibody response depended on the dose of horse antiserum administered and was greater the sooner the serum therapy was applied after envenomation. 3. Injection of antiserum into envenomed mice that received an unrelated antigen (KLH) did not suppress the antibody response to KLH antigen though it inhibited production of antivenom antibodies. 4. Envenomed mice receiving an equivalent dose of F(ab')2 fragments obtained by pepsin digestion of horse antiserum presented the same extent of suppression of the antivenom antibody response as mice injected with the non-treated antiserum. 5. Mice whose antibody response was suppressed, when rechallenged with venom, presented a primary antibody response. 6. These results suggest that suppression of the antivenom antibody response presented by envenomed patients submitted to serum therapy is due to the masking of the venom epitopes by horse antibodies as well as to the rapid elimination of the venom epitopes

Neutralization potency of Russell's viper venom toxoid antivenom, as compared with standard antivenom.

Rabbits were immunized with gamma (gamma) irradiated Russell's viper venom toxoid, adsorbed to aluminium phosphate adjuvant. Antibody (raised against toxoid inoculation) titer was compared to a commercial antivenom on the basis of its ability to neutralize hemorrhagic, necrotic and lethal effects of viper venom. Toxoid immunization (on day 0, 15 and 30) produced antivenom which showed approximately one-third antilethal, half antihemorrhagic and antinecrotic titers as compared to those of the commercial hyperimmunized, concentrated horse antivenom.

Immunization of horses with Crotalus durissus Terrificus (South American Rattlesnake) venom: a comparision of four different procedures

A comparative study was carried out on horses immunized with Crotalus durissus terrificus venom using four different inoculation procedures, which included the use Freund's adjuvant, A1(OH)3 and liposomes as adjuvants. The antibody titer was assessed by enzyme linked immunosorbent assay (ELISA) and the neutralizing potency by the neutralizing median effective dose (ED50). The inoculation schedule used in horses to obtain antivenom serum consisted of scinjections of a 7.5 mg venom starting dose in 5.0ml sterile saline emulsified with an equal volume sterile saline at 2-dayintervals. This immunization procedure, based in low doses of antigen (37.5mg/horse) emulsified with Freund's adjuvant, proceduced a more protective andsustained immune response whencompared with other procedures using A1(OH)3 and 5.0 mg/horse in liposome) or high (870.0 mg/horse in A1(OH)3 and 20.0 mg/horse in liposome) antigen doses. The ED50 values evaluated at the end of the procedure were 15.4 *l serum/20 gmouse when antigen was emulsified with Freund's adjuvant; 21.7 * serum/20 g mouse when 870,0 mg antigen/horse was emulsified with A1(OH)3 and 30.0 *l serum/20 g mouse when 50.0 mg antigen/hors was emulsified with a1(OH)3.When antigen was emulsified with liposome, the immune serum was ineffective against the lethal effects of C.d terrificus venom. The inoculation schedule used in horses to obtain hyperimmune serum consisted of reimmunization with sc booster injections of 7.5 mg venom in 5.0 ml sterile saline emulsified with an equal volume of Freund's incomplete adjuvant. One week later, 2.5 mg venom in 12.0 ml sterile saline was inoculated at 2-day intervals.This reimmunization schedule,based on low doses of antigen (15.0 mg/horse) emulsified with Freund's incomplete adjuvant or with saline, produced a protective andsustained immune response, regardless of the initial immunization procedure. The ED50 evaluatedfor each of the animals five days after the reimmunization period was never more than 20 * serum/20 g mouse. The liposome inoculation method employed a membrane-stabilized reverse phase evaporation preparation of sphingomyelin/cholesterol 2.5/1 (w/w) liposomes. This procedure permits incorporation of 1.0 mg protein per mg of phospholipid. This liposome inoculation method , which stimulates a rapid, sustained and protective immune response in mice and rabbits inoculated with both C.d. collineatus and C.d. terrificus, was not effective when horses were immunized with C.

Neutralization of coral snake Micrurus nigrocinctus venom by a monovalent antivenom

The neutralizing ability of a monovalent anti-Micrurus nigrocinctus (coral snake) antivenom produced in Costa Rica was tested against the letal, myotoxic and phospholipase A, activities of homologous venom. In addition, immunodiffusion and Western blot analyses were performed. in experiments where venom and antivenom were incubated prior to the test, antivenom was effective in neutralizing lethal, myotoxic and phospholipase A2, activities, with Effective Doses 50% of 2700 *l antivenom/mg venom, 1840 *l antivenom/mg venom, and 3630 *l antivenom, respectively. When coral snake antivenom was administered different times after coral snake venom injection, neutralization of lethality was achieved ehen antivenom was injected iv immediately and 15 min after venom. In contrast, lethaly was not reduced when antivenom was administered by the route. Only partial neutralization of myotoxixity was observed even when antivenom was injected iv immediately after envenomation. Immunodiffusion and immunoblot analyses demonstrated the presence of antibodies in antivenom against several, but not all, venom components

Immunological cross reactivity & paraspecificity of the scorpion Heterometrus bengalensis antivenom.

Immunological cross-reactivity and paraspecificity of scorpion H. bengalensis antivenom were studied to find out the intergeneric therapeutic relationship between the venom of other scorpions in West Bengal Buthus tamulus, Lychas laevifrons and Heterometrus swammerdami. Of these scorpions, Buthus tamulus and Lychas laevifrons failed to show any cross reactivity. However, H. swammerdami venom showed cross-reactivity with H. bengalensis antiserum as revealed from immunogeldiffusion and immunoelectrophoresis. This antiserum protected H. swammerdami venom-induced lethality in mice, blocked the contractile response in smooth muscles and antagonised the venom-induced neuromuscular blockade in rat phrenic nerve diaphragm and chick biventer cervicis.

Pharmacological and immunological evaluation of scorpion (Heterometrus bengalensis) venom antiserum.

An antiserum was prepared for the first time against the venom of a common scorpion, H. bengalensis, by hyperimmunization of rabbit. This antiserum showed positive precipitin bands in immunogeldiffusion and immunoelectrophoresis. The serum showed a high titre value tested by indirect haemagglutination test. The antiserum developed in rabbit protected mice against the lethal action of the venom. Smooth muscle contractile response of venom on guinea pig ileum, and rat uterus was antagonized by the antiserum. This antiserum effectively antagonized the venom induced neuromuscular paralysis tested on rat phrenic nerve diaphragm and chick biventer cervices. Antiserum also protected the venom-induced cardiac arrest tested on isolated guineapig heart and auricle preparations.