THE IMPROVEMENT AND STANDARDIZATION OF ANTIVENOM PRODUCTION IN DEVELOPING COUNTRIES: COMPARING ANTIVENOM QUALITY, THERAPEUTICAL EFFICIENCY, AND COST

Abstract

The first antivenom was prepared by Calmette in 1894. More than a century later, it is still the only specific treatment for envenoming. The methods currently used by almost all antivenom producers worldwide to isolate and concentrate antivenom antibodies and their enzymatically derived fragments are improvements of those originally developed by Pope in 1938. Several new alternatives have been proposed to produce F(ab')2 or Fab antivenoms to improve their purity, neutralizing potency, and safety and to overcome the problems encountered in the production protocols based on ammonium sulfate precipitation of equine immunoglobulin. These include complete or partial modifications in the antivenom production regarding animal producers (ovine, laying hens...), immunization protocols, crude serum preparation and/or purification procedures concerning antibody extraction (PEG, caprylic acid, ion-exchange chromatography or immunoaffinity chromatography), and cleavage conditions (pepsin, papain...). In Tunisia, antivenom has been produced since the 1950's. Constant improvements and standardization of all the steps involved in antivenom production, purification, and quality control have resulted in a pure, safe, and efficient F(ab')2 product with no side effects when used intravenously, and with a high seroneutralization yield, as a result of the high toxin specific F(ab')2 concentration. The real impact of the new or modified procedures is a substantial increase in the cost of an antivenom dose. The quality is similar to that of the old procedure when its production was accurately standardized, optimized, correctly conducted and controlled. In addition, severe side effects have been reported after application of either equine F(ab')2 or ovine Fab antivenoms purified by the new methods (i.e. ion-exchange chromatography and immunoaffinity chromatography). Consequently, the introduction of these methods in developing countries still needs justification.

antivenom; standardization of antivenom; F(ab')2 and Fab antivenoms; anaphylactic reaction; anaphylactoid reaction; snake bite; scorpion sting


Review article

THE IMPROVEMENT AND STANDARDIZATION OF ANTIVENOM PRODUCTION IN DEVELOPING COUNTRIES: COMPARING ANTIVENOM QUALITY, THERAPEUTICAL EFFICIENCY, AND COST.

M. N. KRIFI CORRESPONDENCE TO: M. N. KRIFI - Laboratoire de Purification des Immunsérums Thérapeutiques - Institut Pasteur de Tunis 13, Place Pasteur. BP 74; 1002 Tunis-Belvedere. Tunisie - Phone: 216-1-783 022; Fax: 216-1-791 833. , M. EL AYEB , K. DELLAGI

1 Laboratoire de Purification des Immunsérums Thérapeutiques, Institut Pasteur de Tunis. 13, Place Pasteur. BP 74; 1002 Tunis-Belvedere. Tunisie; 2 Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, and 3 Laboratoire d'Immunologie, Institut Pasteur de Tunis.

ABSTRACT: The first antivenom was prepared by Calmette in 1894. More than a century later, it is still the only specific treatment for envenoming. The methods currently used by almost all antivenom producers worldwide to isolate and concentrate antivenom antibodies and their enzymatically derived fragments are improvements of those originally developed by Pope in 1938. Several new alternatives have been proposed to produce F(ab')2 or Fab antivenoms to improve their purity, neutralizing potency, and safety and to overcome the problems encountered in the production protocols based on ammonium sulfate precipitation of equine immunoglobulin. These include complete or partial modifications in the antivenom production regarding animal producers (ovine, laying hens...), immunization protocols, crude serum preparation and/or purification procedures concerning antibody extraction (PEG, caprylic acid, ion-exchange chromatography or immunoaffinity chromatography), and cleavage conditions (pepsin, papain...). In Tunisia, antivenom has been produced since the 1950's. Constant improvements and standardization of all the steps involved in antivenom production, purification, and quality control have resulted in a pure, safe, and efficient F(ab')2 product with no side effects when used intravenously, and with a high seroneutralization yield, as a result of the high toxin specific F(ab')2 concentration. The real impact of the new or modified procedures is a substantial increase in the cost of an antivenom dose. The quality is similar to that of the old procedure when its production was accurately standardized, optimized, correctly conducted and controlled. In addition, severe side effects have been reported after application of either equine F(ab')2 or ovine Fab antivenoms purified by the new methods (i.e. ion-exchange chromatography and immunoaffinity chromatography). Consequently, the introduction of these methods in developing countries still needs justification.

KEY WORDS: antivenom, standardization of antivenom, F(ab')2 and Fab antivenoms, anaphylactic reaction, anaphylactoid reaction, snake bite, scorpion sting.

INTRODUCTION

The first antivenom for human use was prepared by Calmette in 1894 (7,8) based on earlier studies by Von Behring and Kitasato (5) relating to the treatment of diphtheria and tetanus. This and other early antisera were solutions of unrefined horse sera that caused serious reactions. The literature published between 1910 and 1930 indicates that these early antivenoms were sometimes as dangerous as the venom itself (63). Through the intervening years, a number of improvements in antivenom production have been made (62). Nowadays, most antivenoms are either partially purified immunoglobulin (IgG), such as produced in the United States or antigen-binding fragments (F(ab')2), such as produced in Europe and other parts of the world(12).

The methods currently used by almost all antivenom producers worldwide (74) to isolate and concentrate antivenom antibodies are based on salt precipitation with or without enzyme digestion and heat denaturation. These methods are modifications of those originally developed by Pope (50-52), Harms (28), and Pope and Stevens (53) for the purification and concentration of antitoxins and were later applied to snake antivenom (10,20,27,41). These are based on the assumption that the albumin, rather than the globulin fraction, may be responsible for most of the undesirable side effects. However, the use of antivenom prepared as a purified IgG is associated with a high degree of hypersensitivity (12). Rawat et al. (55) reported that the envenoming by the eastern coral snake (Micrurus fulvius fulvius) was treated using IV administration of an equine freeze-dried IgG "Antivenin, Micrurus fulvius" (DRUG CIRCULAR, Wyeth, 1983). Thirty-five percent of the treated patients experienced side effects; 50% of these being severe, resulting in anaphylactic shock or serum sickness (37). Consroe et al. (12) also reported that the only commercially available antivenom for North American Crotalidae is Antivenin (Crotalidae) Polyvalent (equine origin) (ACP; Wyeth Laboratories, Philadelphia, PA). This antivenom may be associated with as many as 15% immediate horse serum reaction and 75% delayed horse serum response (13,61).

To overcome the side effects the majority of producers introduced an enzymatic digestion step in their antivenom purification procedures to split the Fc portion of the IgG molecule that is thought to be responsible for allergic side effects. Smith et al. (67) reported that the current therapy for Vipera berus (common adder) envenoming in U.K., Europe, and Scandinavia is the IV administration of the "European viper antivenom" produced in Zagreb, Croatia. This antivenom, which has an excellent reputation, is a refined F(ab')2 fragment derived from horse serum. However, although lacking the Fc portion, aberrant reactions are seen in 6% to 7% of patients treated with this antivenom. Recently, Moran et al. (46) reported that 76% of the patients intravenously infused with 40-100 ml of the polyvalent F(ab')2 snake antivenom from the South African Institute for Medical Research (SAIMR) developed early severe (anaphylactoid) reactions. Previously published rates of early reactions to this antivenom ranged from < 1% (11,75) to 40% (78).

Some authors (66) explain these reactions by the fact that the administration of F(ab')2 often causes a type III immune-complex mediated hypersensitivity reaction, probably due to the bivalency of this molecule and the finding that immunized horses have high levels of circulating IgGT (18). Thus, IgGT provides most of the protection (19), but it is highly glucosylated and more immunogenic than IgG (66). Morais et al. (45) reported that immune complexes formed between Bothrops venom components and specific antibodies present in the IgG or F(ab')2 antivenoms were able to activate the C complement system. Although immune complexes containing IgG activate equally well the C complement system through both classical and alternative pathways (86.0 and 83.5% of C consumption, respectively), those immune complexes containing essentially F(ab')2 were more effective in activating the alternative than the classical pathway (66.9 and 35.4% of C consumption, respectively). The authors did not evaluate the degree of purity of the F(ab')2 preparation by SDS-PAGE to confirm that it did not contain undigested IgG which could be responsible for this effect rather than F(ab')2. On the other hand, Malasit et al. (43) reported that although antivenom activated complement in vitro, there was no evidence of complement activation or formation of immune complexes in patients bitten by snakes who were treated with antivenom, whether or not they developed early reactions.

The side effects observed after the administration of F(ab')2 as antivenom preparations (6,13,43,44,46,56,67,71,72) seemed to be proportional to the dose of antivenom and the speed with which it entered the blood stream (43). They could also be explained by a poor purity of these preparations as a results of the absence of standardization in the different F(ab')2 purification steps, especially the optimization of either the precipitation (with the complete elimination of the other serum proteins) or the enzymatic digestion of IgG which must result in a complete removal of the Fc portion. The working conditions (temperature, stirring, handling, duration...) could also lead to an important protein denaturation and the appearance of insoluble protein aggregates of high molecular weights, as shown in Ipser-Europe antivenom (49). Antivenom-containing aggregates could activate serum complement and induce deleterious effects in vivo (59). Side-effects can be expected after the administration of Zagreb antivenom, since as reported in a recent paper (42), its purity evaluated by electrophoretic analyses on cellulose polyacetate strips and on sodium dodecylsulfate-polyacrylamide gel showed the presence of almost all the normal equine plasma proteins (high and low molecular weights).

The purity of the currently available horse F(ab')2 preparation against the venoms of Vipera aspis, Vipera ammodytes, and Vipera berus from Ipser Europe (Pasteur-Mérieux, Sérums et Vaccins, France), as published recently (26,49), is not different from that of Zagreb. Ipser Europe antivenom contains only 82% of the polyvalent F(ab')2 and 5.5% of the half-molecule Fab', however the percentages of contaminant proteins of both high (200,000-1,000,000) and low (< 30,000) molecular weights are respectively equal to 4.4 % and 8.1%. Ismail (31) reported that 1.7% to 6.6% of patients treated with F(ab')2 equine scorpion antivenoms (LABS 50, Pasteur-Mérieux Laboratories) developed early reactions. Bucher et al. (6) also observed that 8.1% of the patients experienced early reactions or serum sickness following intravenous infusion of a purified monospecific F(ab')2 Bothrops lanceolatus antivenom manufactured by Pasteur-Mérieux. Compared to the antivenom preparation, the currently available horse F(ab')2 against tetanus toxin (SATP) produced by Pasteur-Mérieux seems to be of a lower purity, since it contains a lower percentage of F(ab')2 (77.4%) and higher percentages of contaminant proteins of low (12.3%) and high (5.9%) molecular weights (49).

Several new alternatives have been proposed including complete or partial modifications in antivenom production or purification procedures to improve the quality of the antivenom and to overcome the problems encountered in the production protocols based on ammonium sulfate precipitation of equine immunoglobulin with or without pepsin digestion. Caprylic acid (17,60), ion-exchange chromatography (48,49), and immunoaffinity (70) chromatography have been proposed to produce equine F(ab')2 or IgG antivenom. Ovine (goat or sheep) Fab antivenoms purified either by using Na2SO4 salt precipitation and papain digestion only (1,39,55,65,66), or by using salt precipitation, papain digestion, and immunoaffinity chromatography (12,64,67) have been also evaluated for their suitability as alternatives to the equine F(ab')2 antivenom. Thalley and Carroll (73) and Carroll et al. (9) described an avian source of rattlesnake and scorpion antivenoms consisting of PEG extraction and immunoaffinity purification of IgY from egg yolks of immunized laying hens.

The new procedure adopted by Pasteur-Mérieux for the purification of horse F(ab')2 scorpion antivenom consisting of two steps of anion-exchange chromatography, pepsin digestion, and heat pasteurization has not significantly improved the purity of F(ab')2 preparation compared to that purified by the old procedure based on ammonium sulfate precipitation. As reported recently by Pepin-Covatta et al., (48,49), the purity of the new F(ab')2 preparation was 88%. Contaminants were mainly low molecular weight (<20 kDa) components (3%). The remaining percentages were Fab' (5% to 7%) and a molecular species of 200 kDa (1% to 2%). Polymers and aggregates accounted for 0.6%. In addition, for viper antivenom, only 33% of the total venom protein of molecular weights 17,500, 28,500, 32,000, and 60,000 seem to be recognized by the new F(ab')2 preparations as determined by western blot of electrophoresis gels. The minor improvement induced by the Pasteur-Mérieux F(ab')2 new purification procedure raises more questions than proposes real solutions to the problems encountered with the old F(ab')2 purification procedure. In addition, Meyer et al. (44) reported that 5% of envenomed patients developed early (anaphylactic) reactions after injection of Pasteur-Mérieux IPSER Africa antivenom; an equine polyspecific (Bitis-Echis-Naja) F(ab')2 preparation.

The use of affinity purified specific Fab has a real advantage, especially for the treatment of scorpion envenoming in regard to its specificity, pharmacokinetics, and the feature of the toxicokinetics of scorpion venom toxic proteins related to the smallness of their molecular weight. However, the safety and efficacy of Fab preparations in humans must be validated before they can be widely used. Dart and Horowitz (15) reported that three patients envenomed by Vipera berus and treated with ovine Fab antivenom developed acute allergic reactions and serum sickness. Meyer et al. (44) observed that the injection of one vial (10 ml, 0.5 g proteins) or two vials (20 ml, 1 g protein) of monospecific E. ocellatus antivenom consisting of an ovine Fab fragment (Echi Tab; Therapeutic antibodies, Ltd.) caused early anaphylactic reactions respectively, in 23% and 57% of the cases. The cost of the affinity purified Fab, the upkeeping of the immunosorbent column, which must be always pyrogen free, and the total elimination of the papain from the final product, in addition to the problems related to the chromatographic procedure itself could be other serious drawbacks of this antivenom production procedure.

With regard to antivenoms from hen eggs, Landon et al. (40) reported that avian proteins are notoriously allergenic and their use could be associated with a high incidence of side effects. The affinity purification step will result in a more specific product, but in time, it will certainly be accompanied by a significant increase in antivenom price.

It is also interesting to know if these new purification procedures have the same yield* * (Yield = potency (LD50/ml) of crude sera x total volume of crude sera x 100) potency of purified sera x total volume of purified sera in terms of seroneutralization as the old ones. Are the additional chromatographic steps (anion-exchange or affinity) accompanied by any improvement or any decrease in the yield of the antivenom purification procedure? What is the percentage increase in the cost per antivenom dose related to these new or modified procedures? Is the resulting quality improvement of the F(ab')2 or Fab antivenom preparation sufficient to justify the related increase in cost? Chromatographic purification procedures show several important drawbacks. For example, the purification of IgG directly from whole plasma or after a first step of ammonium sulfate precipitation (14), and the separation of F(ab')2 from undigested IgG and pepsic digested proteins by anion-exchange chromatography needed several intermediate steps, such as equilibration in chromatography buffer, dialysis or desalting steps, elution of IgG, desorption of adsorbed plasma proteins, which generally needed a change in the ionic strength of the buffer, and finally a re-equilibration step before re-use of the column. For a good separation, the elution must be performed at a very low flow rate, which makes this technique time consuming and could result in bacterial contamination of the product, causing an increase in the pyrogenicity of the antivenom. The elution and the desalting steps dilute the product (a dilution factor of 1.5-2.5 was reported by Corthier et al., (14); thus, a step of concentration seems to be necessary.

Sjostrom et al. (66) proposed that the ideal antivenom for use in developing countries, where envenoming is most common must be safe, effective, with no side effects when administered systemically, and inexpensive. Biochemically speaking, the best antivenom is that which shows the highest purity, potency, and the lowest protein content (79). Medically speaking, the best antivenom is that showing the highest therapeutic efficiency with the lowest dose and no side effects. Economically speaking, the best antivenom is that exhibiting the best balance between therapeutic efficiency and cost. The most sophisticated purification procedure is not always necessary to give the best balance between antivenom quality, yield, and production cost.

Safety and efficiency (efficacy) of envenoming immunotherapy are especially and closely related, respectively, to the purity of the antivenom and the accurate determination of its neutralizing potency. These two properties of a given antivenom greatly influence its therapeutic use in envenomings. Although all antivenom producers worldwide use basically the same method to purify and cleave equine immunoglobulins (or not) enzymatically to produce F(ab')2 or Fab fragments, the results of this purification procedure in terms of purity, final potency protein content, and yield could be very different from one producer to another. For example, the antivenom produced by Wyeth contains 70% to 90% IgG and 5% to 25% albumin as the main contaminants (15,66,69). See also Pepin-Covatta et al. (48,49), Lomonte et al. (42), and Meyer et al. (44) for respectively Pasteur-Mérieux, Zagreb, and Therapeutic antibodies, Ltd. antivenom products. These antivenom preparations can be expected to produce unacceptable rates of acute and delayed complications. Moreover, Ismail (30-32) reported that the analysis of imported antivenom available against local scorpion venoms gave a potency of only 20% to 40% of that stated on the label.

In Tunisia antivenom has been produced at the Pasteur Institute since the 1950's. Constant improvements and standardization of all steps of antivenom production, purification, and quality control have resulted in a pure, safe, and efficient F(ab')2 product free from albumin, undigested IgG, high molecular weight protein, and aggregates as evaluated by SDS-PAGE 12.5% (Figure 1) and electrophoresis on cellulose acetate (Figure 2). This improved purification procedure has a good seroneutralization yield of 65%. For anti-scorpion venom production, horses are immunized only with the toxic fractions purified from a pool of crude venom by using gel filtration chromatography on Sephadex-G50, which results in a good immunoreactivity of the F(ab')2 as determined by western blot of SDS-PAGE 12.5% gel electrophoresis. The concentration of specific F(ab')2 assessed by immunoaffinity chromatography varied from 7.93 to 11.74 mg/ml, which represents 6.9% to 10.20% of the total F(ab')2. Al-Asmari et al. (1) reported that the concentration of specific antibodies directed against W. aegyptia venom in Behringwerke and Pasteur-Mérieux antivenoms was only 3 and 2 mg/ml, respectively. The venom used for antivenom sera production and potency assessment is a large pool from more than 10,000 individual milkings. Scorpions are collected in all endemic areas to avoid geographical venom variations. The antivenom is bivalent since horses are immunized with the toxic fractions of Androctonus australis and Buthus occitanus venoms. However, because of the high antigenic similarity between venoms of scorpions of the Buthidae family (16,57,58), the antivenom is also efficient against the venoms of Androctonus aeneas and Androctonus mauritanicus mauritanicus, as assessed in the laboratory. This antivenom also neutralizes by paraspecificity the venom of other scorpions from the same family, such as the Leiurus quinquestriatus (3,4,16,24,25,29,36,54,77). In addition, it seems that polyvalent or bivalent antivenoms have a better paraspecificity neutralization than monovalent antivenoms (25). The Tunisian improved antivenom is largely used as a specific scorpion or viper envenoming immunotherapy, especially intravenously (20-80 ml). As reported by clinicians in charge of scorpion and viper envenomed patients, adverse reactions were rarely observed, and when they occurred they were local and very mild. These results may be explained because scorpion venoms are known to induce the release of tissue and medullary catecholamines (2,21-23,30-35,47,68) that protect the patients from early anaphylactic reactions. However, the high purity of F(ab')2, the absence of undigested IgG and protein aggregates of a high molecular weight, the high percentage of specific F(ab')2, and the good balance between the total protein content and the neutralizing potency could also explain the absence of anaphylactic reactions.

FIGURE 1.
SDS-PAGE analysis of F(ab')2 antivenoms. SDS-PAGE was performed according to Laemmli (1970) (38). Samples were boiled for 5 min in 4% SDS, then applied to a polyacrylamide gel (12.5%). The gel was stained with Coomassie Brilliant Blue R250. Protein molecular weight (MW) markers were: myosin (212kDa), a2-macroglobulin (170 kDa), b-galactosidase (116 kDa), phosphorylase b (94 kDa), transferrin (76 kda), albumin (67 kDa), glutamic dehydrogenase (53 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20,1 kDa), and a-lactalbumin (14 kDa). Lanes 1 and 6: Marker proteins. Lanes 2 to 5: Electrophoretic pattern of four purified F(ab')2 antivenom batches.
FIGURE 2.
Electrophoretic analysis of scorpion antivenoms on cellulose polyacetate. A: Equine crude serum. B: Purified F(ab')2 antivenom. The anode is on the left. 1: Albumin, 2: a1-a2, 3: b and 4: g globulins

On the other hand, an overview of the literature seems to indicate that side effects have been more frequently reported after snake antivenom therapy than after scorpion antivenom therapy. However, antivenom is still the only specific treatment, and in cases of severe envenoming, evidence has shown that the benefits of antivenom therapy (44,46,76) outweigh the risks of side effects (76). We also think that anaphylactic reactions are better controlled than severe envenoming outcomes.

In developing countries, where scorpion and snake envenomings are most common, it is clearly evident that it is possible to produce high quality inexpensive antivenom using only the optimized, standardized, and adequately controlled old preparation procedure. However, this antivenom preparation procedure must meet national and international GMP requirements, as well as the WHO, US Pharmacopoeia, and European Pharmacopoeia recommendations.

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41 LATIFI M., MANHOURI H. Antivenin production. Ann. Inst. Butantan, 1966, 33, 893-8.

42 LOMONTE B., LEON G., HANSON LA. Similar effectiveness of Fab and F(ab')2 antivenoms in the neutralization of hemorrhagic activity of Vipera berus snake venom in mice. Toxicon, 1996, 34, 1197-202.

43 MALASIT P., WARRELL DA., CHANTHAVANICH P., VIRAVAN C., MONGKOLSAPAYA J., SINGHTHONG B., SUPIGH C. Prediction, prevention and mechanism of early (anaphylactic) antivenom reactions in victims of snakes bites. Br. Med. J., 1986, 292, 17-20.

44 MEYER WP., HABIB AG., ONAYADE AA., YAKUBU A., SMITH DC., NASIDI A., DAUDU IJ., WARRELL DA., THEAKSTON RDG. First clinical experiences with a new ovine Fab Echis ocellatus snake bite antivenom in Nigeria: randomized comparative trial with Institute Pasteur serum (IPSER) Africa antivenom. Am. J. Trop. Med. Hyg., 1997, 56, 291-300.

45 MORAIS JF., DE FREITAS MCW., YAMAGUCHI IK., DOS SANTOS MC., DIAS DA SILVA W. Snake antivenoms from hyperimmunized horses: comparison of the antivenom activity and biological properties of their whole IgG and F(ab')2 fragments. Toxicon, 1994, 32, 724-34.

46 MORAN NF., NEWMAN WJ., THEAKSTON RDG., WARRELL DA., WILKINSON D. High incidence of early anaphylactoid reaction to SAIMR polyvalent snake antivenom. Trans. R. Soc. Trop. Med. Hyg., 1998, 92, 69-70.

47 PATTERSON RA. Physiological action of scorpion venom. Am. J. Trop. Med. Hyg., 1960, 9, 410-4.

48 PEPIN-COVATTA S., LUTSCH C., GRANDGEORGE M., LANG J., SCHERRMANN, JM. Immunoreactivity and pharmacokinetics of horse anti-scorpion venom F(ab')2-scorpion venom interactions. Toxicol. Appl. Pharmacol., 1996, 141, 272-7.

49 PEPIN-COVATTA S., LUTSCH C., GRANDGEORGE M., LANG J., SCHERRMANN, JM. Immunoreactivity of a new generation of horse F(ab')2 preparations against European viper venoms and the tetanus toxin. Toxicon, 1997, 35, 411-22.

50 POPE CG. Desegregation of proteins by enzymes. Br. J. Exp. Pathol., 1938, 19, 245-51.

51 POPE CG. The action of proteolytic enzymes on the antitoxins and proteins in immune sera. I. True digestion of the proteins. Br. J. Exp. Pathol., 1939, 20, 132-49.

52 POPE CG. The action of proteolytic enzymes on the antitoxins and proteins in immune sera. II. Heat denaturation after partial enzyme action. Br. J. Exp. Pathol., 1939, 20, 201-12.

53 POPE CG., STEVENS MG. The action of proteolytic enzymes on the antitoxins and proteins in immune sera. III. Further studies on enzyme systems which split the antitoxin molecule. Br. J. Exp. Pathol., 1951, 32, 314-24.

54 POTTER JM., NORTHEY WT. An immunological evaluation of scorpion venoms. Am. J. Trop. Med. Hyg., 1962, 11, 712-6.

55 RAWAT S. LAING G., SMITH DC., THEAKSTON D., LANDON J. A new antivenom to treat eastern coral snake (Micrurus fulvius fulvius ) envenoming. Toxicon, 1994, 32, 185-90.

56 REID HA. Antivenom reactions and efficacy. Lancet, 1980, 1, 1024-5.

57 ROCHAT H., ROCHAT C., KOPEYAN C., MIRANDA F., LISSITZKY S., EDMAN P. Scorpion neurotoxins: a family of homologous proteins. FEBS Letter, 1970, 10, 349.

58 ROCHAT H., BERNARD P., COUROUD F. Scorpion toxins: chemistry and mode of action. Adv. Cytopharmacol., 1979, 3, 325-34.

59 ROJAS G., ESPINOZA M., LOMONTE B., GUTIERREZ JM. Effect of storage temperature on the stability of the liquid polyvalent antivenom produced in Costa Rica. Toxicon, 1990, 28, 101-5.

60 ROJAS G., JIMENEZ JM., GUTIERREZ JM. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon, 1994, 32, 351-63.

61 RUSSELL FE. Snake venom poisoning. Great Neck: Scholium International, 1983: 235-343.

62 RUSSELL FE. Snake venom immunology: historical and practical considerations. J. Toxicol. Toxin Rev., 1989, 7, 1-82.

63 RUSSELL FE., SCHARFENBERG RS. Bibliography of snake venoms and venomous snakes. West Covina: Bibliographic Associates, 1962: 27p.

64 RUSSELL FE., SULLIVAN JB., EGEN NB., JETER WS., MARKLAND FS., WINGERT WA., BAR-OR D. Preparation of a new antivenin by affinity chromatography. Am. J. Trop. Med. Hyg., 1985, 34, 141-50.

65 SELLS PG., JONES RGA., LAING GD., SMITH DC., THEAKSTON RDG. Experimental evaluation of ovine antisera to Thai cobra (Naja kaouthia) venom and its a-neurotoxins. Toxicon, 1994, 32, 1657-65.

66 SJOSTROM L., AL ABDULLA IH., RAWAT S., SMITH DC., LANDON J. A comparison of ovine and equine antivenoms. Toxicon, 1994, 32, 427-33.

67 SMITH DC., REDDI KR., LAING G., THEAKSTON RDG., LANDON J. An affinity purified ovine antivenom for the treatment of Vipera berus envenoming. Toxicon, 1992, 30, 865-71.

68 STHANKE HL. Some pharmacological and biomedical characteristics of Centruroides sculpturatus (Ewing) scorpion venom. Rec. Adv. Pharmacol. Toxins, 1965, 9, 63-70.

69 SULLIVAN JR JB. Past, present and future immunotherapy of snake venom poisoning. Ann. Emerg. Med., 1987, 16, 938-44.

70 SULLIVAN JR JB., RUSSELL FE. Isolation and purification of antibodies to rattlesnake venom by affinity purification. Proc. West. Pharmacol. Soc., 1982, 25, 185-9.

71 SUTHERLAND SK. Serum reactions. An analysis of commercial antivenoms and a possible role of anticomplementary activity in de novo reactions of antivenoms and toxins. Med. J. Aust., 1977, 1, 613-5.

72 SUTHERLAND SK., LOVERING KE. Antivenoms: use and adverse reactions over a 12-month period in Australia and Papua New Guinea. Med. J. Aust., 1979, 2, 671-4.

73 THALLEY BS., CARROLL SB. Rattlesnake and scorpion antivenoms from the egg yolks of immunized hens. Bio/Technology, 1990, 8, 934-8.

74 THEAKSTON RDG., WARRELL DA. Antivenoms: a list of hyperimmune sera currently available for the treatment of envenoming by bites and stings. Toxicon, 1991, 29, 1419-70.

75 VISSER J., CHAPMAN DS. Snakes and snakebites. Johannesburg: Centaur Publishers, 1978: 92-6.

76 WARRELL DA. Management of snakebite. In: WEATHERALL DJ., LEDINGHAM JGG., WARRELL DA. Eds. Oxford textbook of Medicine. 3.ed. Oxford: Oxford University Press, 1996: 1135-40.

77 WHITTEMORE JR FW., KEEGAN HL., BOROWITZ JL. Studies of scorpion antivenins. I. Paraspecificity. Bull. World Health Organ., 1961, 25, 185-8.

78 WILKINSON D. Retrospective analysis of snakebite at a rural hospital in Zululand. S. Afr. Med. J., 1994, 84, 844-7.

79 WHO. Normes relatives aux immunsèrums d'origine animale. WHOSér. Rapp. Tech., 1969, 413, 47-61.

Received 12 August 1998

Accepted 06 October 1998

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  • 02 AMARAL CFS., DIAS MB., CAMPOLINA D., PROIETTI FA., REZENDE NA. Children with adrenergic manifestations of envenomation after Tityus serrulatus scorpion sting are protected from early anaphylactic antivenom reaction. Toxicon, 1994, 32, 211-5.
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  • 10
    CHRISTENSEN PA. The preparation and purification of antivenoms. Mem. Inst. Butantan, 1966, 22, 245-50.
  • 11
    CHRISTENSEN PA. Snakebite and the use of antivenom in Southern Africa. S. Afr. Med. J., 1981, 59, 934-8.
  • 12
    CONSROE P., EGEN NB., RUSSELL FE., GERRISH K. SMITH DC., SIDKI A., LANDON JT. Comparison of a new ovine antigen binding fragment (Fab) antivenin for United States Crotalidae with the commercial antivenin for protection against venom-induced lethality in mice. Am. J. Trop. Med. Hyg, 1995, 53, 507-10.
  • 13
    CORRIGAN P., RUSSELL FE., WAINSCHEL J. Clinical reactions to antivenin. In: ROSENBERG, P. Ed. Toxins: animal, plant and microbial. Oxford: Pergamon Press, 1978: 457-65.
  • 14
    CORTHIER G., BOSCHETTI E., CHARLEY-POULAIN J. Improved method for IgG purification from various animal species by ion exchange chromatography. J. Immunol. Methods, 1984, 66, 75-9.
  • 15
    DART RC., HOROWITZ RS.Use of antibodies as antivenoms: a primitive solution for a complex problem.In: BON, C., GOYFFON, M. Eds. Envenomings and their treatments Lyon : Fondation Marcel Mèrieux, 1996: 83-94.
  • 16
    DELORI P., VAN RIETSCHOTEN J., ROCHAT H. Scorpion venoms and neurotoxins: an immunological study. Toxicon, 1981, 19, 393-407.
  • 17
    DOS SANTOS MC., D'IMPERIO LIMA MR., FURTADO GC., COLLETTO GMDD., KIPNIS T.L., DIAS DA SILVA W. Purification of F(ab')2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield. Toxicon, 1989, 27, 297-303.
  • 18
    EK N. Serum levels of the immunoglobulins IgG and IgG(T) in horses. Acta Vet. Scand., 1974, 15, 609-19.
  • 19
    FERNANDES I., TAKEHARA HA., MOTA I. Isolation of IgGT from hyperimmune horse anti-snake venom serum: its protective ability. Toxicon, 1991, 29, 1373-9.
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    FREIRE-MAIA L., PINTO GI., FRANCO I. Mechanism of the cardiovascular effects produced by purified scorpion toxin in the rat. J. Pharmac. Exp. Ther.,1974, 188, 207-13.
  • 23
    GHAZAL A., ISMAIL M., ABDEL-RAHMAN AA., EL-ASMAR MF. Pharmacological studies of scorpion (Androctonus amoreuxi, Aud and Sav.) venom. Toxicon, 1975, 13, 253-9.
  • 24
    GLENN WG., KEEGAN HL., WHITTEMORE FWJr. Intergeneric relationships among various scorpion venins and antivenins. Science, 1962, 135, 434-5.
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    GOYFFON M. Scorpionisme et sérums antiscorpioniques. Rev. Arachnol., 1984, 5, 311-9.
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    GRANDGEORGE M., VERON JL., LUTSCH C., MAKULA MF., RIFFARD P., PEPIN S., SCHERRMANN JM. Preparation of improved F(ab')2 antivenoms. An example: new polyvalent European viper antivenom (equine). In: BON, C., GOYFFON, M. Eds. Envenomings and their treatments. Lyon: Fondation Marcel Mérieux, 1996: 161-72.
  • 27
    GRASSET E., CHRISTENSEN PA. Enzyme purification of polyvalent antivenin, South and Equatorial African colubrine and viperine venoms. Trans. R. Soc. Trop. Med. Hyg., 1947, 41, 207-11.
  • 28
    HARMS AJ. The purification of antitoxic plasma by enzyme treatment and heat denaturation. Biochem. J., 1948, 42, 390-7.
  • 29
    IRUNBERRY J., PILO-MORON E. Etude antigénique de quelques venins de scorpions du bassin Mèditerranèen. Arch. Inst. Pasteur Algér., 1965, 43, 123-8.
  • 30
    ISMAIL M. Serotherapy of the scorpion envenoming syndrome is irrationally convicted without trial. Toxicon, 1993, 31, 1077-83.
  • 31
    ISMAIL M. The treatment of the scorpion envenoming syndrome. The Saudi experience with serotherapy. Toxicon, 1994, 32, 1019-26.
  • 32
    ISMAIL M. The scorpion envenoming syndrome. Toxicon, 1995, 33, 825-58.
  • 33
    ISMAIL M., OSMON OH., IBRAHIM SA., EL-ASMAR MF. Cardiovascular and respiratory responses to the venom from the scorpion Leiurus quinquestriatus. S. Afr. Med. J, 1972, 49, 273-81.
  • 34
    ISMAIL M., OSMON OH., IBRAHIM SA., EL-ASMAR MF. Pharmacological studies of the venom from the scorpion Buthus minax (L. Koch).Toxicon, 1973, 11, 15-20.
  • 35
    ISMAIL M., OSMON OH., GUMAA KA., KARRARA, MA. Some pharmacological studies with the scorpion (Pandinus exitialis) venom.Toxicon,1974, 12, 75-82.
  • 36
    JUNQUA C., VACHON M. Les arachnides venimeux et leurs venins. Etat actuel des recherches. Acad. R. Sci. Outre-Mer. Cl. Sci. Nat., 1968, 17, 1-136.
  • 37
    KITCHENS CS., VAN MIEROP LHS. Envenomation by the eastern coral snake Microbus fulvius fulvius, a study of 39 victims. J. Am. Med. Ass, 1987, 258, 1615-8.
  • 38
    LAEMMLI UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227, 680-5.
  • 39
    LAING GD., LEE L., SMITH DC., LANDON J., THEAKSTON RDG. Experimental assessment of a new low-cost antivenom for treatment of carpet viper (Echis ocellatus) envenoming. Toxicon, 1995, 33, 307-13.
  • 40
    LANDON J., WOOLLEY JA., McLEAN C. Antibody production in the hen. In: LANDON, J., CHARD T. Ed. Therapeutic antibodies. Berlin: Springer Verlag, 1995: 47-68.
  • 41
    LATIFI M., MANHOURI H. Antivenin production. Ann. Inst. Butantan, 1966, 33, 893-8.
  • 42
    LOMONTE B., LEON G., HANSON LA. Similar effectiveness of Fab and F(ab')2 antivenoms in the neutralization of hemorrhagic activity of Vipera berus snake venom in mice. Toxicon, 1996, 34, 1197-202.
  • 43
    MALASIT P., WARRELL DA., CHANTHAVANICH P., VIRAVAN C., MONGKOLSAPAYA J., SINGHTHONG B., SUPIGH C. Prediction, prevention and mechanism of early (anaphylactic) antivenom reactions in victims of snakes bites. Br. Med. J., 1986, 292, 17-20.
  • 44
    MEYER WP., HABIB AG., ONAYADE AA., YAKUBU A., SMITH DC., NASIDI A., DAUDU IJ., WARRELL DA., THEAKSTON RDG. First clinical experiences with a new ovine Fab Echis ocellatus snake bite antivenom in Nigeria: randomized comparative trial with Institute Pasteur serum (IPSER) Africa antivenom. Am. J. Trop. Med. Hyg., 1997, 56, 291-300.
  • 45
    MORAIS JF., DE FREITAS MCW., YAMAGUCHI IK., DOS SANTOS MC., DIAS DA SILVA W. Snake antivenoms from hyperimmunized horses: comparison of the antivenom activity and biological properties of their whole IgG and F(ab')2 fragments. Toxicon, 1994, 32, 724-34.
  • 46
    MORAN NF., NEWMAN WJ., THEAKSTON RDG., WARRELL DA., WILKINSON D. High incidence of early anaphylactoid reaction to SAIMR polyvalent snake antivenom. Trans. R. Soc. Trop. Med. Hyg., 1998, 92, 69-70.
  • 47
    PATTERSON RA. Physiological action of scorpion venom. Am. J. Trop. Med. Hyg., 1960, 9, 410-4.
  • 48
    PEPIN-COVATTA S., LUTSCH C., GRANDGEORGE M., LANG J., SCHERRMANN, JM. Immunoreactivity and pharmacokinetics of horse anti-scorpion venom F(ab')2-scorpion venom interactions. Toxicol. Appl. Pharmacol., 1996, 141, 272-7.
  • 49
    PEPIN-COVATTA S., LUTSCH C., GRANDGEORGE M., LANG J., SCHERRMANN, JM. Immunoreactivity of a new generation of horse F(ab')2 preparations against European viper venoms and the tetanus toxin. Toxicon, 1997, 35, 411-22.
  • 50
    POPE CG. Desegregation of proteins by enzymes. Br. J. Exp. Pathol., 1938, 19, 245-51.
  • 51
    POPE CG. The action of proteolytic enzymes on the antitoxins and proteins in immune sera. I. True digestion of the proteins. Br. J. Exp. Pathol., 1939, 20, 132-49.
  • 52
    POPE CG. The action of proteolytic enzymes on the antitoxins and proteins in immune sera. II. Heat denaturation after partial enzyme action. Br. J. Exp. Pathol., 1939, 20, 201-12.
  • 53
    POPE CG., STEVENS MG. The action of proteolytic enzymes on the antitoxins and proteins in immune sera. III. Further studies on enzyme systems which split the antitoxin molecule. Br. J. Exp. Pathol., 1951, 32, 314-24.
  • 54
    POTTER JM., NORTHEY WT. An immunological evaluation of scorpion venoms. Am. J. Trop. Med. Hyg., 1962, 11, 712-6.
  • 55
    RAWAT S. LAING G., SMITH DC., THEAKSTON D., LANDON J. A new antivenom to treat eastern coral snake (Micrurus fulvius fulvius ) envenoming. Toxicon, 1994, 32, 185-90.
  • 56
    REID HA. Antivenom reactions and efficacy. Lancet, 1980, 1, 1024-5.
  • 57
    ROCHAT H., ROCHAT C., KOPEYAN C., MIRANDA F., LISSITZKY S., EDMAN P. Scorpion neurotoxins: a family of homologous proteins. FEBS Letter, 1970, 10, 349.
  • 58
    ROCHAT H., BERNARD P., COUROUD F. Scorpion toxins: chemistry and mode of action. Adv. Cytopharmacol., 1979, 3, 325-34.
  • 59
    ROJAS G., ESPINOZA M., LOMONTE B., GUTIERREZ JM. Effect of storage temperature on the stability of the liquid polyvalent antivenom produced in Costa Rica. Toxicon, 1990, 28, 101-5.
  • 60
    ROJAS G., JIMENEZ JM., GUTIERREZ JM. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon, 1994, 32, 351-63.
  • 61
    RUSSELL FE. Snake venom poisoning. Great Neck: Scholium International, 1983: 235-343.
  • 62
    RUSSELL FE. Snake venom immunology: historical and practical considerations. J. Toxicol. Toxin Rev., 1989, 7, 1-82.
  • 63
    RUSSELL FE., SCHARFENBERG RS. Bibliography of snake venoms and venomous snakes. West Covina: Bibliographic Associates, 1962: 27p. 
  • 64
    RUSSELL FE., SULLIVAN JB., EGEN NB., JETER WS., MARKLAND FS., WINGERT WA., BAR-OR D. Preparation of a new antivenin by affinity chromatography. Am. J. Trop. Med. Hyg., 1985, 34, 141-50. 
  • 65
    SELLS PG., JONES RGA., LAING GD., SMITH DC., THEAKSTON RDG. Experimental evaluation of ovine antisera to Thai cobra (Naja kaouthia) venom and its a-neurotoxins. Toxicon, 1994, 32, 1657-65.
  • 66
    SJOSTROM L., AL ABDULLA IH., RAWAT S., SMITH DC., LANDON J. A comparison of ovine and equine antivenoms. Toxicon, 1994, 32, 427-33.
  • 67
    SMITH DC., REDDI KR., LAING G., THEAKSTON RDG., LANDON J. An affinity purified ovine antivenom for the treatment of Vipera berus envenoming. Toxicon, 1992, 30, 865-71.
  • 68
    STHANKE HL. Some pharmacological and biomedical characteristics of Centruroides sculpturatus (Ewing) scorpion venom. Rec. Adv. Pharmacol. Toxins, 1965, 9, 63-70.
  • 69
    SULLIVAN JR JB. Past, present and future immunotherapy of snake venom poisoning. Ann. Emerg. Med., 1987, 16, 938-44.
  • 70
    SULLIVAN JR JB., RUSSELL FE. Isolation and purification of antibodies to rattlesnake venom by affinity purification. Proc. West. Pharmacol. Soc., 1982, 25, 185-9.
  • 71
    SUTHERLAND SK. Serum reactions. An analysis of commercial antivenoms and a possible role of anticomplementary activity in de novo reactions of antivenoms and toxins. Med. J. Aust., 1977, 1, 613-5.
  • 72
    SUTHERLAND SK., LOVERING KE. Antivenoms: use and adverse reactions over a 12-month period in Australia and Papua New Guinea. Med. J. Aust., 1979, 2, 671-4.
  • 73
    THALLEY BS., CARROLL SB. Rattlesnake and scorpion antivenoms from the egg yolks of immunized hens. Bio/Technology, 1990, 8, 934-8.
  • 74
    THEAKSTON RDG., WARRELL DA. Antivenoms: a list of hyperimmune sera currently available for the treatment of envenoming by bites and stings. Toxicon, 1991, 29, 1419-70.
  • 75
    VISSER J., CHAPMAN DS. Snakes and snakebites. Johannesburg: Centaur Publishers, 1978: 92-6.
  • 76
    WARRELL DA. Management of snakebite. In: WEATHERALL DJ., LEDINGHAM JGG., WARRELL DA. Eds. Oxford textbook of Medicine. 3.ed. Oxford: Oxford University Press, 1996: 1135-40.
  • 77
    WHITTEMORE JR FW., KEEGAN HL., BOROWITZ JL. Studies of scorpion antivenins. I. Paraspecificity. Bull. World Health Organ., 1961, 25, 185-8.
  • 78
    WILKINSON D. Retrospective analysis of snakebite at a rural hospital in Zululand. S. Afr. Med. J., 1994, 84, 844-7.
  • 79
    WHO. Normes relatives aux immunsèrums d'origine animale. WHOSér. Rapp. Tech., 1969, 413, 47-61.

  • CORRESPONDENCE TO:
    M. N. KRIFI - Laboratoire de Purification des Immunsérums Thérapeutiques - Institut Pasteur de Tunis 13, Place Pasteur. BP 74; 1002 Tunis-Belvedere. Tunisie - Phone: 216-1-783 022; Fax: 216-1-791 833.
  • *
    (Yield =
    potency (LD50/ml) of crude sera x total volume of crude sera x 100)
    potency of purified sera
    x total volume of purified sera

Publication Dates

  • Publication in this collection
    17 Sept 1999
  • Date of issue
    1999

History

  • Received
    12 Aug 1998
  • Accepted
    06 Oct 1998
Centro de Estudos de Venenos e Animais Peçonhentos - CEVAP, Universidade Estadual Paulista - UNESP Caixa Postal 577, 18618-000 Botucatu SP Brazil, Tel. / Fax: +55 14 3814-5555 | 3814-5446 | 3811-7241 - Botucatu - SP - Brazil
E-mail: jvat@cevap.org.br