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Redalyc.Use of Hen Egg Derived Immunoglobulin Against Scolopendra Revista Científica ISSN: 0798-2259 [email protected] Universidad del Zulia Venezuela Parrilla-Alvarez, Pedro; Navarrete, Luis F.; Girón, María E.; Aguilar, Irma; Rodríguez-Acosta, Alexis Use of hen egg derived immunoglobulin against scolopendra (Scolopendra gigantea) venom Revista Científica, vol. XVIII, núm. 4, julio-agosto, 2008, pp. 385-392 Universidad del Zulia Maracaibo, Venezuela Disponible en: http://www.redalyc.org/articulo.oa?id=95918407 Cómo citar el artículo Número completo Sistema de Información Científica Más información del artículo Red de Revistas Científicas de América Latina, el Caribe, España y Portugal Página de la revista en redalyc.org Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto _______________________________________________________Revista Científica, FCV-LUZ / Vol. XVIII, Nº 4, 385 - 392, 2008 USE OF HEN EGG DERIVED IMMUNOGLOBULIN AGAINST SCOLOPENDRA (Scolopendra gigantea) VENOM Uso de Inmunoglobulina Derivada de Huevo de Gallina contra el Veneno de Escolopendra (Scolopendra gigantea) Pedro Parrilla-Alvarez1.2, Luis F. Navarrete2, María E. Girón2, Irma Aguilar2 and Alexis Rodríguez-Acosta2* Laboratorio de Alacranología, Facultad de Medicina, Universidad de Oriente. Ciudad Bolívar, Venezuela. Sección de Inmunoquímica, Instituto de Medicina Tropical de la Universidad Central de Venezuela. Caracas, Venezuela. E-mail: rodriguezacosta1946@ yahoo.es RESUMEN ABSTRACT Las picaduras de escolopendra (Scolopendra gigantea)ense- A scolopendra sting in humans and domestic animals is an res humanos y animales domésticos representan un accidente acute and highly painful accident. The present study was an at- agudo y muy doloroso. La necrosis y otros daños ocasionados tempt to raise specific hyper-immune polyclonal antibodies por este veneno pueden ser prevenidos, si se inyecta un anti- against scolopendra centipede (Scolopendra gigantea Lin- veneno. Este estudio propone producir anticuerpos policlona- neaus 1758) venom. A group of hens were injected with ven- les en gallinas hiper-inmunizadas contra el veneno de escolo- oms subcutaneously and intramuscularly according to three pendra (Scolopendra gigantea Linneaus 1758). Un grupo de different routines. This protocol was found to be effective for gallinas fue inyectado subcutánea e intramuscularmente con hyperimmunization. Eggs were gathered daily and antibodies diluciones de venenos, de acuerdo con tres rutinas diferentes. were purified from yolk with a polyethylene-glycol and chloro- Los huevos fueron recogidos diariamente y los anticuerpos en form modified method. Titers of antibodies in yolk were esti- la yema fueron purificados con un método modificado de polie- mated with an agar gel precipitation test, and a serum protec- tilen-glicol y cloroformo. Los niveles de anticuerpos en yema tion (ED#) test. Eggs harvested at 15 days post-injection had fueron calculados con prueba de precipitación de gel de agar y maximum antibody titers. After six months, antibodies lyophi- pruebas de protección (ED#). Los huevos cosechados 15 días lized and stored at 5°C still maintained their activity. Enven- post-inyección tenían los títulos más altos de anticuerpos. omed mice were injected with purified antibodies, which in- Después de seis meses, los anticuerpos liofilizados y guarda- duced 100% recovery as compared to those not treated with dos a 5°C mantenían su actividad. Ratones envenenados, in- the antibodies. The cleanliness, effectiveness, and simplicity of yectados posteriormente con anticuerpos purificados, tuvieron producing antibodies against scolopendra venom in avian egg un 100% de supervivencia al compararse con los controles. La yolk, and their incapability to attach human complement, for- limpieza, la eficacia, y la sencillez de producir los antivenenos mulates an interesting option to equine and other mammalian en gallina, y la incapacidad de estos anticuerpos (IgY) para fi- antivenoms. This study infers that avian egg yolk antibodies jarse al complemento humano, formulan una alternativa intere- may be useful as a therapeutic tool in treating scolopendrism in sante a otros antivenenos producidos en mamíferos. Este es- humans and domestic animals. It also opens a field for the pro- tudio puntualiza que los anticuerpos de huevo de gallina pue- duction of other antivenoms against the wide spectrum of tox- den ser provechosos como un instrumento terapéutico para ins as well as the use of these antibodies as a diagnostic tool. tratar escolopendrismo en seres humanos y animales domésti- cos. Además, abre un campo terapéutico para la fabricación Key words: Immunoglobulin Y, scolopendra antivenom, scolo- de otros antivenenos contra el espectro amplio de las toxinas y pendrism, Scolopendra gigantea. como probable herramienta de diagnóstico. Palabras clave: Inmunoglobulina Y, antiveneno de escolopen- INTRODUCTION dra, escolopendrismo, Scolopendra gigantea. Scolopendrism is the envenomation by species of Scolopendra [12]. The venom is toxic to humans (the venom Recibido: 19 / 03 / 2007. Aceptado: 02 / 10 / 2007. contains serotonin, acetylcholine, histamine, lipids, polysac- 385 Use of hen egg derived immunoglobulin against Scolopendra gigantea venom / Parrilla-Alvarez, P. y col.____________________________ charides, enzymes such as proteases, hyaluronidases and es- glands. They were anesthetized by maintaining them at –20°C terases [1,12] and causes a local painful burn (similar to a for approximately 10 min. The specimens were fixed by means hornet-sting), severe edema, chills, fever (up to 39°), weak- of two connected stimulating electrodes (Grass model, USA). ness, erythema, local lymphangitis, dizziness, necrosis at the An electrode was placed in the body and the other one in the site of the sting, myonecrosis and acute renal damage [1,17]. base of the claw. Once the animal was fixed, an electric stimu- At experimental levels the Scolopendra venom causes, accord- lation was carried out by administering pulses of 60V per pulse ing to the dose, death in mice (Mus musculus) within a few at a frequency of 50 Hz to each claw. Each claw was given 3 to minutes [31]. Accidents are not rare in human adults and se- 5 pulses for approximately 15 s, with resting periods of ap- vere envenoming followed by death is an exception. Neverthe- proximately 30 s. Ejected venom through the excretory conduit less, some fatal cases have been reported [20]. When the in the tip of the claw was collected in glass capillaries that were scolopendra is irritated, it inoculates its venom through claws placed in Eppendorf tubes and stored at –30°C until use. The or fangs; each gland drains its toxic contents through a small volume of each extraction was calculated knowing the volume aperture near the tip of the fang, and drops into the wounds of the capillary used in the collection. causing the referred symptoms. In order to avoid venom contamination with gastric juices Passive immunization with antibodies derived mainly and also avoid the capillary fracture, special mask containing from horse’s (Equus caballus) blood is widely used to treat holes to accommodate each claw and render them immobile snake (Viperidae), scorpion (Buthidae), and spiders (Arachnoi- were used. dea) envenomations [17]. However, there is no specific treat- ment offered for scolopendra envenomation and the production Determination of protein concentration of antivenom is necessary for its treatment. The crude venom was resuspended in distilled water and The current study was intended to produce and purify the protein concentration was determined by means of absor- hen (Gallus domesticus) specific hyper-immune polyclonal an- bance read in a spectrometer (Spectronic® Model Genesis 2, tibodies against scolopendra venom. Evaluation of antibodies USA) at 280 nm, assuming that 1 absorbance unit represented was carried out through agar gel precipitation and serum pro- a concentration of 1 mg/mL. tection (ED50) tests, in order to evaluate the antivenom as an efficient therapeutic agent. Lethality assay The intraperitoneal LD50 for scolopendra crude venom MATERIALS AND METHODS was determined in mice. Three of 10 mice (18-22 g) each were injected intraperitoneally with four doses of venom (0.3 µg to Animals 0.6 µg in 0.1 mL) dissolved in saline. Forty eight hours later Scolopendra. A group (twelve specimens) of Scolopen- dead animals were recorded and the results analyzed by the dra gigantea, coming from Zulia and Falcon States, Venezuela, Spearman-Karber method [27]. were maintained in the Immunochemistry Section of the Insti- tute of Tropical Medicine at the Universidad Central de Vene- Sodium dodecyl sulphate-polyacrylamide gel zuela. The animals, once identified, were placed in boxes and electrophoresis (SDS-PAGE) of scolopendra crude venom fed monthly with arthropods or neonates mice. Scolopendra crude venom under reduced and non re- Mice. Male mice (INH strain) weighing 18-22 g from the duced conditions was electrophoresed using a MINI- Instituto Nacional de Higiene “Rafael Rangel”, Caracas, PROTEAN II (BioRad, USA) chamber. SDS-PAGE was per- Venezuela were used. The colony of mice was kept in boxes formed according to the Laemmli method [15] using 12% gels. in a room maintained at 23°C on a 12/12-hr light/dark cycle as Wide range molecular weight markers (Bio-Rad) were run in outlined in the “Guide of Principles of Laboratory Animal parallel and gels were stained with Coomassie Blue R-250. Care” [3]. Hens. Six egg-laying, red hens of Rhode Island strain of Immunization of egg-laying
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