Abstracts

INTRODUCTION

Snake envenomation produces tissue effects such as local swelling and necrosis, neurotoxicity and hemostatic disorders. Considerable advancement has been made in understanding the pathophysiology of ophidic accidents, motivating transformations in treatment procedures. Latest advances, including the production of new antivenoms using new processes¹1. Almeida CM, Kanashiro MM, Rangel Filho FB, Mata MF, Kipnis TL, da Silva WD. Development of snake antivenom antibodies in chickens and their purification from yolk. Vet Rec. 1998;143:579-84.–⁴4. Chippaux JP, Williams V, White J. Snake venom variability methods of study, results and interpretation. Toxicon. 1991;29:1279-303.,¹⁹19. Parrilla P, Navarrete LF, Girón M.E, Aguilar I, Rodríguez-Acosta A. Use of chicken egg yolk-derived immunoglobulin against Scolopendra venom as an alternative to treat scolopendrism. Rev Cient FCV-LUZ. 2008;18:385-92., have encouraged developing coral snake antivenoms with attractive protocols.

Nearly two hundred species from the Elapidae family are dispersed across the Southeastern and Southwestern United States, as well as all of Mexico, Central America and South America, and are also established in the African, Asian and Oceanic continents³3. Campbell JA, Lamar WW. The venomous reptiles of Latin America. Ithaca: Cornell University Press; 1989.. On the American continent, more than 120 species and subspecies have been described, separated into three genera: Leptomicrurus with three species, Micruroides, with one, and Micrurus, with approximately 70 species²¹21. Prieto da Silva AR, Yamagushi IK, Morais JF, Higashi HG, Raw I, Ho PL, et al. Cross reactivity of different specific Micrurus antivenom sera with homologous and heterologous snake venoms. Toxicon. 2001;39:949-53.,²²22. Roze JA. Coral snakes of the Americans. Biology, identification, and venoms. Florida: Krieger Publishing Co; 1996..

The production of safe, efficient and reasonably priced antivenoms is a priority. Alternative progress in the therapeutics of coral snake bite victims in Venezuela requires an answer to the production of new animal models, logistical, financial, marketing, delivery and storage difficulties related to the supply of antivenom. Furthermore, the norms and regulations for physicians to improve antivenom use must also be addressed. Even though coral snake envenomations could be handled using medications that act on presynaptic and postsynaptic receptors, since some patients may only survive under intensive therapy treatment such as respiratory support²³23. Sánchez EE, Lopez-Johnston JC, Rodríguez-Acosta A, Pérez JC. Neutralization of two North American coral snake venoms with United States and Mexican antivenoms. Toxicon. 2008;51:297-303., specific treatment with antivenoms continues to be the elected method for treating these incidents, which can efficiently deactivate all systemic activities of the venom. Nevertheless, there are some collateral effects of antivenom such as anaphylaxis and serum sickness⁵5. Christensen PA. Production and standardization of antivenin. In: Lee CY, editor. Handbook of experimental pharmacology. New York: Springer-Verlag; 1979. p. 1-825.. The majority of these alterations seem to be caused by the action of high concentrated proteins, which are not immunoglobulins, but contaminating polyvalent antivenoms. However, the benefits of antivenom treatment may be more important than its risks.

The specific therapeutics for coral snake envenomations is the use of heterologous antivenom, and to date, this type of antivenom is not available in Venezuela. In light of the information that coral snake venom can reveal a multiplicity of composition and toxic activities, we have included the most important venom species occurring in Venezuela and United States in the immunization protocol. At this time, we present a study on the production of a specific coral snake (Micrurus) antivenom and its purification of immunoglobulins from the egg yolk of immunized hens, with the purpose of providing a more efficient antivenom for therapeutic treatments.

MATERIALS AND METHODS

Ethical statement

All the experimental events concerning the use of live animals were carried out by specialized staff. The relevant regulations of Venezuela as well as institutional guidelines, according to protocols approved by the Institute of Anatomy of the Universidad Central de Venezuela, and the norms obtained from the guidelines for the care and use of laboratory animals¹³13. Institute of Laboratory Animal Resources. Guide for the care and use of laboratory animals. 8th ed. Washington: National Research Council, National Academies Press; 2011. were followed.

Coral snakes (Micrurus) venom

Venom from a collection (twenty specimens) of different Micrurus species from Venezuela and the United States were used in the immunization protocol. The venom of Venezuelan coral snakes consisted of (Micrurus isozonus (Calabozo, Guárico State), Micrurus isozonus (Caracas, D.C), Micrurus isozonus (La Boyera, Miranda State), and Micrurus isozonus (Maracay, Aragua State) which were supplied by the Serpentarium of the Tropical Medicine Institute of the Universidad Central de Venezuela, Caracas, Venezuela. The venom from the U.S. consisted of Micrurus fulvius fulvius(Eastern, USA), and Micrurus tener tener(Western, USA), purchased from the National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, Texas, USA.

Fifteen days prior to venom extraction, the coral snakes were fed and made to fast to guarantee enough venom in their glands. The venom was collected through a 50-mL plastic centrifuge tube transversely cut and covered on the top with Parafilm® (Millipore Corp, USA). The snake was forced to bite the Parafilm. Venom was collected by glass capillaries through the excretory conduit in the base of the fangs, centrifuged, and supernatants were placed in Eppendorf® (Eppendorf Int, USA) tubes and stored at -30 °C until use. Stock solutions were prepared in phosphate-buffered saline (PBS) (10 Mm sodium phosphate containing 150 Mm NaCl, Ph 7.2) at 1.0 mg/mL.

Mice

Female mice (INH strain) weighing 18-20 g were obtained from the Instituto Nacional de Higiene “Rafael Rangel”, Caracas, Venezuela. The colony of mice was kept in plastic boxes (Tecniplast, Italy) at six mice per cage, in a room maintained at 23 °C on a 12/12-hr light/dark cycle.

Hens

Six egg-laying, red hens (Gallus domesticus) of Rhode Island strain, approximately 16 weeks old, obtained from a poultry farm from Lagunita town, Miranda State, Venezuela, were located in individual henhouses (Centenosuplidores C.A, Venezuela)(one hen per henhouse) prior to the commencement of the production of eggs. Hens were maintained on 12/12-hr light/dark cycle at 23 °C with food and water ad libitum.

Determination of protein concentration

Protein determination was established by the method of LOWRY et al.¹⁵15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ, Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-75..

Micrurus' venom lethality

Lethality of crude venom was determined by intravenous injections into mice and the LD₅₀ value calculated according to the Spearman-Karber method³¹31. World Health Organization. Progress in the characterization of venoms and standardization of antivenoms. Who Offset Publ. 1981;58:1-44.. The venom was diluted in a phosphate-buffered saline solution (PBS). The endpoint of lethality of the mice was determined after 48h. All solutions during the experiments were stored at 4 °C and warmed to 37 °C prior to being injected into mice. The lethal toxicity was determined in five groups containing five mice. A total of 0.2 mL of venom (dosages from 0.05 to 0.8 mg/kg) was injected into the tail vein of 18-20 g female BALB/c mice. A comparable volume of PBS was injected as a negative control group.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of coral snake's crude venom

Pools of different coral snakes' crude venom under non-reduced conditions were electrophoresed using a MINIPROTEAN II (BioRad, USA) chamber. SDS-PAGE was performed according to the Laemmli method (1970)¹⁴14. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-5.using 15% gels. Wide range molecular weight markers (Bio-Rad) were run in parallel and gels were stained with Comassie blue (National Diagnostic, USA).

Immunization

A pool was made with concentrations of venom, corresponding to the LD₅₀ median. A sub-lethal dose was used for immunizations. Four-month-old egg-laying hens, weighing ∼1 kg were maintained pathogen-free and immunized with the pool of coral snakes' venom. Venom (0.24 mg/kg in 0.1 mL) was taken into an Eppendorf tube and then mixed with an equal volume of Freund's complete adjuvant, whereas the second doses consisted of venom emulsified with Freund's incomplete adjuvant (GIBCO, USA). The third venom doses were mixed with a saline solution. All doses were administered subcutaneously, via the deltoid region in four different places, alternating right and left every two weeks for eight weeks. One week after the last dose, the hens' blood was obtained for the detection of immunoglobulins that could recognize and precipitate the coral snake venom.

Isolation of immunoglobulin

The modified method of SVENDSON et al.(1995)³⁰30. Svendson L, Crowley A, Ostergaard LH, Stodulski G, Hau J. Development and comparison of purification strategies for chicken antibodies from egg yolk. Lab Anim Sci. 1995;45:89-3.using the freeze and thaw principle to remove lipids was used. For a brief period, the egg yolk was diluted 10 times with distilled water, and the diluted egg yolk was frozen at -80 °C overnight, and permitted to thaw at an increased temperature rate of ∼ 2 °C (six min) by maintaining it at 2-4 °C. The egg yolk was then centrifuged at 18,000 g for 1h at 4 °C and the supernatant gathered was cleared by filtration on Whatman (N°. 1) filter paper (Whatman, USA). To precipitate the immunoglobulins, the filtered supernatant was then precipitated by 40% ammonium sulfate at 4 °C. Afterwards, the pellet was re-suspended in 0.01M Tris-HCl (pH 8.0) to a volume equal to half of the supernatant. Following centrifugation for 15 min at 13 000 g, the pellet was washed twice with 40% ammonium sulfate (ReAgent, UK). The solution was dialyzed carefully (three buffer changes, at least 150 times volume) against 10 mM phosphate buffer, pH 7.0, for 24 to 48h in a dialyzing tube with molecular cut off weight of 14 KDa to remove the ammonium sulfate⁵5. Christensen PA. Production and standardization of antivenin. In: Lee CY, editor. Handbook of experimental pharmacology. New York: Springer-Verlag; 1979. p. 1-825.,¹⁹19. Parrilla P, Navarrete LF, Girón M.E, Aguilar I, Rodríguez-Acosta A. Use of chicken egg yolk-derived immunoglobulin against Scolopendra venom as an alternative to treat scolopendrism. Rev Cient FCV-LUZ. 2008;18:385-92..

Antibody activity and purity were determined using a SDS-PAGE¹⁴14. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-5., double gel diffusion test¹⁸18. Ouchterlony O. Diffusion-in-gel methods for immunological analysis. Prog Allergy. 1958;5:1-78. and a serum protection test (ED₅₀), respectively.

Gel diffusion assay using specific IgY against coral snake venom

To show the specific IgY immunoglobulin activity, a 1% agarose (Sigma-Aldrich Co. USA) double gel diffusion test was used¹⁸18. Ouchterlony O. Diffusion-in-gel methods for immunological analysis. Prog Allergy. 1958;5:1-78.. The immunizing pool of coral snake crude venom at 10 mg/mL was placed in the central well, while different coral snake venoms and PBS were placed in the outer wells and incubated at 37 °C.

Serum protection test (Median effective dose = ED₅₀)

For antivenom effectiveness, five groups of eight mice were challenged with a mixture of varying concentrations of IgY antivenom containing three LD₅₀ of venom. The antivenom/venom mixture was prepared at 0 °C and incubated for 30 min at 37 °C prior to injection. Each mouse was injected with 0.2 mL of venom/antivenom mixture into the tail vein. The mice were observed for 48 h and the survival percentage and ED₅₀ were calculated according to Spearman and Karber³¹31. World Health Organization. Progress in the characterization of venoms and standardization of antivenoms. Who Offset Publ. 1981;58:1-44.. Saline controls and antivenom controls were used. Neutralizing capability was expressed as the 50% effective dose (the amount of antivenom that protects 50% of the population) (Table 2).

Specificity of coral snake antivenom (IgY) to various snake venom via Western blot

To explore the specificity of the immunoglobulins against coral snake venom, the antibodies were also assayed with Crotalus and Bothrops venom. A total of nine venom (22 µg/each) consisting of M. isozonus, M. surinamensis, M. f. fulvius, M. t. tener, Naja kaouthia, Naja pallida, Bothrops colombiensis, Crotalus durissus cumanensis and C. vegrandis were electrophoresed on a 10-20% Tricine SDS gel using a XCell SureLock^™ system at 150V (Bio-Rad PowerPac Basic) for one hour. The proteins were transferred onto a 0.2 µm nitrocellulose membrane (Millipore) using a Trans Blot SD system (BioRad) at 100 mA for one hour. The primary antibody (chicken anti-coral snake venom IgY) was diluted to 1/200, the secondary antibody (rabbit anti-chicken IgY-alkaline phosphatase) (Sigma-Aldrich Co. USA) was diluted to 1/50,000. SIGMA FAST^™ BCIP/NBT tablets were used to visualize the bands on the blot and SimplyBlue (Life Technologies, USA) was used to visualize the bands on the gel. SeeBluePlus2 (Life Technologies, USA) markers were used as standards.

Antivenom

The antivenom passed the standard tests for neutralizing potency, miocrobiological purity, lack of pyrogenicity, appropriate protein concentration, lack of abnormal toxicity, sterility, and pH (6.9)¹¹11. Gutiérrez JM, Rojas E, Quesada L, Léon G, Núñez J, Laing GD, et al. Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa. Trans R Soc Trop Med Hyg. 2005;99:468-75..

RESULTS

Lethality assay

The LD₅₀ calculated as a median from the mixture of coral snake venom used for immunization was 0.58 mg/kg. The LD₅₀values were different among Micrurus venoms tested, with LD₅₀ of 0.32 mg/kg (Micrurus fulvius), 0.5 mg/kg (Micrurus isozonus) and 0.78 mg/kg (Micrurus tener) (Table 1).

Immunization

The LD₅₀s for the venoms were carried out according to the SÁNCHEZ et al.²³23. Sánchez EE, Lopez-Johnston JC, Rodríguez-Acosta A, Pérez JC. Neutralization of two North American coral snake venoms with United States and Mexican antivenoms. Toxicon. 2008;51:297-303. method. A sub-lethal dose of the pool of venoms made with concentrations corresponding to the LD₅₀ median of 0.3 mg/kg, was used to immunize the hens (Table 1).

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of coral snake crude venom

The SDS-PAGE (15%) protein profiles of Micrurus isozonus venoms were analyzed. The individual venom differed in composition, quantity and intensity of bands (Fig. 1). The M. isozonus venom from Calabozo contained more protein bands above 31.kDa (Fig. 1; lane 2) than all other M. isozonus venom. The M. isozonus venom from Caracas and Maracay both contained a similar protein band between 66.2 and 97.4 kDa (Fig. 1; lanes 3 and 5, respectively). The sample from La Boyera (Fig. 1; lane 4) contained all protein bands below 21.5 kDa.

Isolation of immunoglobulin

This method³⁰30. Svendson L, Crowley A, Ostergaard LH, Stodulski G, Hau J. Development and comparison of purification strategies for chicken antibodies from egg yolk. Lab Anim Sci. 1995;45:89-3.involved a gently synchronized -70 °C freeze and 4 °C thaw cycle, consequently giving a clear egg yolk solution. The total protein concentration in one egg yolk was around 1.3 ± 0.5 g. In this method, a larger component of the additional proteins was excluded through lipid elimination of egg yolk by the freeze and thaw cycle. IgY was reduced with β-Mercaptoethanol (Sigma-Aldrich Co. USA) showing a heavy chain fragment of 68 kDa and light chain of 27 kDa (Fig. 3). The material, after precipitation, contained about 90% pure IgY.

Gel diffusion assay using specific IgY against coral snake venom

The existence of antivenom IgY in egg yolk was tested by Ouchterlony's immunodiffusion assay¹⁸18. Ouchterlony O. Diffusion-in-gel methods for immunological analysis. Prog Allergy. 1958;5:1-78. using crude venom as the antigen (Fig. 2). The protein concentration in the wells (1-12) was 1 mg/ mL, and 20 µL was added to each well. Antivenom added to the middle well was 20 µL, containing a concentration of 1 mg/mL Lowry's protein. Several precipitin lines at each location pointed out the polyvalent character of the antibodies. The antibody titer augmented after the booster dose, and antibodies were present 180 days after the first injection.

Serum protection test (Median effective dose = ED₅₀)

Coral snake antivenom (IgY) was able to effectively neutralize the pool of all Micrurus isozonus coral snake venom used in the immunization with a mean ED₅₀ of 477.8 mg/kg (Table 2).

Specificity of coral snake antivenom (IgY) to various snake venom via Western blot

The coral snake antivenom was able to recognize, to some degree, all the coral snake venoms used in this study in addition to two cobra snake venoms and three Venezuelan pit vipers. The antivenom was most specific to M. f. fulvius followed by M. isozonus, M. t. tener and M. surinamensis. The two Naja spp. had similar reactions to each other, and the two Crotalus spp. also showed similarities. Bothrops colombiensis venom had a different reactivity pattern than the other Venezuelan venom. Micrurus surinamensis venom had the least reaction with the antivenom compared to all the venom tested. No protein bands below 12 kDa were detected for any venom.

DISCUSSION

Despite the significant pressure due to death and grave consequences resulting from accidents with coral snakes, the research and investment of funds for the production of antivenoms is considered insufficient. On the other hand, biochemical studies relating to Micrurus venoms are limited, due to the complexity of accurately identifying the species, the difficulties in maintaining them in captivity, and the difficulty in obtaining a desirable amount of venom; in addition, venom varies between intra and interspecies in composition, associated with their age, gender, geographic distribution, and diet⁴4. Chippaux JP, Williams V, White J. Snake venom variability methods of study, results and interpretation. Toxicon. 1991;29:1279-303.,¹⁰10. Gutiérrez JM, Chaves F, Rojas R, Bolaños R. Efectos locales inducidos por el veneno de la serpiente coral Micrurus nigrocinctus en Ratón Blanco. Toxicon. 1980;18:633-9..

In this study, the competence of hens to make antibodies against small quantities of antigens was studied. In some conditions, there may be problems with getting adequate amounts of antigen to make immunizations. In this experiment the hens were immunized with 0.1-100 µg of antigen with booster immunizations. Every two weeks for eight weeks, the IgY response in the yolk demonstrated a similar picture as that in the serum. The study confirmed that it is possible to obtain a good immune response with less amounts of antigen than is usually recommended to immunize sheep or horses. Studies by some groups have established that antibody responses to foreign antigens are genetically controlled. It has been feasible to breed hens that are high and low antibody responders intramuscularly²⁰20. Pinard MH, van Arendonk JA, Nieuwland MG, van der Zijpp AJ. Divergent selection for immune responsiveness in chickens: estimation of realized heritability with an animal model. J Anim Sci. 1992;70:2986-93. or by intravenous⁹9. Gross WG, Siegel PB, Hall RW, Domermuth CH, DuBoise RT. Production and persistence of antibodies in chickens to sheep erythrocytes. 2. Resistance to infectious diseases. Poult Sci. 1980;59:205-10.,¹⁶16. Martin A, Dunnington EA, Gross WB, Briles WE, Briles RW, Siegel PB. Production traits and alloantigen systems in lines of chickens selected for high or low antibody responses to sheep erythrocytes. Poult Sci. 1990;69:871-8. injections.

The production of IgY from small amounts of antigen required for immunization, the ease in collecting the eggs, and the uncomplicated purification techniques to enhance the function of immunological assays, make the use of IgY attractive and profitable.

Normally, antivenoms are achieved by immunizing horses with increasing doses of venom to obtain a high-quality antibody titer⁸8. Estrada R, Chaves F, Robles A, Rojas E, Segura E, Gutiérrez JM. Valores hematológicos y de enzimas séricas en caballos inoculados com venenos de serpientes para la producción de antivenenos en Costa Rica. Rev Biol Trop. 1992;40:95-9.. A range of side effects exists in the administration of antivenom; for instance, anaphylaxis and serum sickness²⁷27. Schellekens H. How to predict and prevent the immunogenicity of therapeutic proteins. Biotechnol Annu Rev. 2008;14:191-202.. The elevated concentrations of proteins, which are not antibodies, existing in many common antivenom probably produce most of these symptoms. With the aim of eliminating the unpleasant effects of the antivenom treatment, it is necessary to achieve antivenom immunoglobulins in a reasonable purification state. Investigators²⁴24. Sells PG, Laing GD, Theakston RD. An in vivo but insensate model for the evaluation of antivenoms (ED50) using fertile hens' eggs. Toxicon. 2001;39:665-8.,²⁵25. Schade R, Calzado EG, Sarmiento R, Chacana PA, Porankiewicz-Asplund J, Terzolo HR. Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine. Altern Lab Anim. 2005;33:129-54.,³⁰30. Svendson L, Crowley A, Ostergaard LH, Stodulski G, Hau J. Development and comparison of purification strategies for chicken antibodies from egg yolk. Lab Anim Sci. 1995;45:89-3. have proposed that birds are a suitable and economical supply of IgY immunoglobulins. Chicken egg yolks have IgY, which is a species-specific immunoglobulin with a molecular weight of 190kDa²⁶26. Schade R, Schniering A, Hlinak A. Polyclonal avian antibodies extracted from egg yolk as an alternative to the production of antibodies in mammals: a review. ALTEX. 1992; 9:43-56.. Our laboratory has produced hen antibodies against Scolopendra gigantea toxins with high titers¹⁹19. Parrilla P, Navarrete LF, Girón M.E, Aguilar I, Rodríguez-Acosta A. Use of chicken egg yolk-derived immunoglobulin against Scolopendra venom as an alternative to treat scolopendrism. Rev Cient FCV-LUZ. 2008;18:385-92.. Others have reported the production of immunoglobulins against different types of protein antigens, synthetic peptides, etc.²⁹29. Stuart CA, Pietrzyk RA, Furlanetto RW, Green A. High affinity antibody from hen's eggs directed against the human insulin receptor and the human IGF-I receptor. Anal Biochem. 1988;173:142-50.. We have improved the production of polyclonal antibodies by means of crude coral snake venom injected in hens with an alternative protocol, from that of the classical method used for horse immunoglobulin production. In our current work, we were capable of obtaining a good antibody titer with immunized hens.

Due to the high incidences of coral snake envenomations by M. isozonus in Venezuela, this particular snake venom was used to carry out the in vivo efficacy test of coral snake IgY antivenom, which resulted in ED₅₀ of 477 mg/kg body weight (Table 2). This value falls within acceptable ED₅₀ ranges of other antivenoms tested on different snake venoms⁷7. Devi CM, Bai MV, Lal AV, Umashankar PR, Krishnan LK. An improved method for isolation of anti-viper venom antibodies from chicken egg yolk. J Biochem Biophys Methods. 2002;51:129-38.. A Western blot was done to determine the specificity of the IgY antivenom to different coral snake venoms as well as other Venezuelan venomous species. It appears that the IgY coral snake venom antivenom was able to recognize all four coral snake venoms used in the immunization protocol with M. isozonus, M. surinamensis, M. f. fulvius and M. t. tener having the highest recognition. The venom of M. surinamensis was the least reactive (Fig. 4). In addition to those venoms used to produce the IgY antivenom, two cobra venoms (N. kaouthia and N. pallida), and three Venezuelan Viperidae species (B. colombiensis, C. d. cumanensis and C. vegrandis) were also detected by the antivenom. This cross reactivity suggests there are similar venom toxins found in species of snakes located in distinct geographical locations. The fact that M. surinamensis had the least reactivity of all venoms, including those not used in the immunization protocol, indicates the uniqueness of the proteins found in that venom. The possible use of IgY in therapy and/or diagnostic assays either in humans or animals provides an invaluable field of interesting and useful applications¹²12. Hernández-Campos FJ, Brito-De la Fuente E, Torrestiana-Sánchez B. Purification of egg yolk immunoglobulin (IgY) by ultrafiltration: effect of pH, ionic strength, and membrane properties. J Agric Food Chem. 2010;58:187-93.,¹⁷17. Meenatchisundaram S, Parameswari G, Michael A, Ramalingam S. Studies on pharmacological effects of Russell's viper and Saw-scaled viper venom and its neutralization by chicken egg yolk antibodies. Int Immunopharmacol. 2008;8:1067-73.,²⁸28. Spillner E, Braren I, Greunke K, Seismann H, Blank S, du Plessis D. Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy. Biologicals. 2012;40:313-22..

Foundation Project: This research was supported by grants from the FONACIT (Venezuela)(G-2005000400), Shell Venezuela S.A, Ministry of the Popular Power for Science and Technology (LOCTI program), CDCH/UCV PG: 09-8760-2013/1, and the Natural Toxins Research Center and Department of Chemistry at Texas A&M University-Kingsville, Kingsville, Texas, USA.

REFERENCES

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