Research Article. Assessment of preclinical efficacy of antivenoms produced in rabbit by immunological methods and neutralization assays

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1 Available online Journal of Chemical and Pharmaceutical Research, 2014, 6(12): Research Article ISSN : CODEN(USA) : JCPRC5 Assessment of preclinical efficacy of antivenoms produced in rabbit by immunological methods and neutralization assays Fatima Zahra Khaddach 2, Brahim Benaji 2,3, Fatima Laraba Djebari 4, Fatima Chgoury 1, Lotfi Boussadda 1, Amina Wadi 1, Naoual Oukkache 1 and Noreddine Ghalim 1 * 1 Venoms and Toxins Laboratory, Pasteur Institute of Morocco, Place Louis Pasteur, Casablanca, Morocco 2 Department of biology, Pharmacology and Toxicology unity, Microbiology-Pharmacology-Biotechnology and Environment Laboratory, Faculty of Sciences Ain Chock, Hassan II University, Maarif, Casablanca, Morocco 3 Ecole Normale Supérieure de l Enseignement Technique de Rabat, Université Mohammed V Souissi, Avenue de l'armée Royale, Madinat Al Irfane, Rabat, Morocco. 4 Laboratoire de Biologie cellulaire et Moléculaire, Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumediene, Alger, Algeria ABSTRACT Envenomation by Moroccan vipers (Cerastes cerastes Cc and Macrovipera mauretanica Mm) has caused significant morbidity and mortality. In Morocco, both vipers were increasingly identified as a dangerous and common source of envenomation. The antivenom production in Morocco against poisonous snakes encounters a number of difficulties; unfortunately, there is still no monospecific Moroccan antivenom to date. This study aims to assess the feasibility of monospecific Cc and Mm antivenom production F(ab ) 2 prepared under the same immunization protocols in rabbits. SDS-PAGE analysis of both types of antivenoms showed similar serum protein profiles. C.cerastes venom elicited satisfactory titers of anti-cc F(ab ) 2 after immunization as compared to M.mauretanica venom. Both antivenoms, isolated with ammonium sulfate precipitation method, were effective in neutralizing the venom lethality (potency = and LD 50 per ml for anti-cc and anti-mm respectively) as well as its hemorrhagic effects induced by 3MHD of the venom. Cross-reactivity studies using Ouchterlony test and indirect ELISA (Enzyme-Linked Immunosorbent Assay) showed that anti-cc and anti-mm F(ab ) 2 cross-reacted extensively with several venoms, particularly that of viper species (Bitis arietans), presumably due to the presence of venom antigens common to both snakes. Keywords: Monospecific antivenom, ELISA, Moroccan vipers, neutralization, potency, Cross-reactivity INTRODUCTION Snake bites are a major public health problem throughout the world and has recently been incorporated by the World Health Organization (WHO) in its list of neglected diseases [1,2], especially in tropical countries where mortality and morbidity rates are very high (i.e., sub-saharan Africa) [3]. Vipers are among the potentially dangerous snakes given their relatively frequent bites in North-Africa [4]. In Morocco, it was reported that 1761 envenomations by venomous snakes occurred between [5]. From these reported cases, 62% were considered to be symptomatic. Lethality was estimated at 7.2%, occurring mainly among rural populations. The total number of envenomation cases in Morocco may be underestimated in so far as there is no reliable data nor is there a systematic snake bite reporting [6]. Cerastes cerastes and Macrovipera mauretanica species are classified among the potentially incriminated in most envenomation accident cases reported in Morocco [7-8]. Although, the discovery of immunotherapy dates as far back as more than a century ago [9], it remains the treatment of choice of ophidian envenomation [10]. In Morocco, snake envenomation faces various pertinent challenges, 446

2 including that of the quality of production and supply of antivenom [11, 2] due to the unavailability of geographically adapted antivenoms. A successful treatment of snakebite envenomation depends on the quality of the administered antivenom. The latter is required to be highly pure and specific to avoid most of the side effects and hypersensitivity reactions caused by foreign proteins [12-14]. Although polyvalent antivenoms is still the only proven therapy for snake envenomations [15-16], there is a belief that they are less effective and cause higher incidence of adverse reactions, such as illness and, severe allergic reactions, a fact which has limited the use of this treatment. The preparation of potent highly pure antivenoms requires many experiments. The aim of such experiments is twofold: to establish optimal immunization conditions and to create the ideal fractionation circumstances to obtain a high yield and purity product in terms of the reduction of the albumin content, which ideally should not exceed 1% of total protein in the final product [16], to avoid the onset of adverse reactions in snake venom treated patients [17-19]. It is, therefore, preferable to use monovalent antivenoms for the treatment of envenomation [10, 20, 21]. These antivenoms are preferred unless the identification of the snake species is uncertain. In such a case, cross-reactions and/or cross-neutralizations become very good assisting factors [10]. Owing to a lack of local production of specific antivenom, most countries in these regions, like Morocco, fully depend on foreign supplies of antivenoms. Often, the effectiveness of the imported antivenoms against local medically important species has not been validated [22]. In Morocco, for example, there is still no specific antivenom to date for the treatment of viper envenomation. The administration of commercial polyclonal antivenom remains the only treatment for snake bites; a previous epidemiological and clinical evaluation of snake bites demonstrated the limitation of the current antivenom treatment. The debates over the relative superiority of monovalent antivenoms and polyvalent antivenoms [23] have suggested that the monovalent antivenoms exhibit better potency and are less likely to cause adverse reactions. The efficiency of such treatment is still controversial, particularly in the case of mild and moderate envenomations and remains strongly dependent on antivenom efficiency complying with the Good Manufacturing Practices recommended by WHO [16]. Venoms of the viperid snakes are generally highly hematoxic and most of the envenoming inflicted by the vipers are often characterized by hemorrhage and tissue necrosis [24, 25], thus complicating the identification of biting species and their clinical management[26]. Based on this information, the aim of this work was to assess the efficiency of monospecific antivenoms, elicited by C.cerastes and M.mauretanica venom, raised in rabbits. It also sought to evaluate the physicochemical properties (e.g. electrophoresis) and immunochemical reactivity, tested by double immunodiffusion and ELISA, of C.cerastes and M.mauretanica antivenoms and ultimately assess the immune cross - neutralization between Cc and Mm antivenoms against other dangerous snakes venom in Morocco, such as Bitis arietans and Naja haje legionis. The present study also investigated, the in vivo venom-neutralizing potencies (Neutralization of lethality and hemorrhage ) of these monospecific immunoglobulins F(ab ) 2. This approach can therefore be used to improve the strategy of producing the most efficient and protective antivenom. EXPERIMENTAL SECTION 1. Materials 1.1. Biological material Animals Male albino Swiss mice weighing between g and male rabbits weighing about 3.5 Kg were used in this study. The animals were housed according to Good Manufacturing Practices guidelines on animal experimentation, and received food and water ad libitum before being used for study. Snake venoms Venoms were milked from adult specimens snakes of C. cerastes, M. mauretanica kept in captivity at the Serpentarium of Pasteur Institute of Morocco at Tit Mellil, Morocco. Venoms were obtained by manual compression of the venom glands. After extraction, pooled venoms were centrifuged, filtered, lyophilized and stored at -20 C until use Reagents Complete Freund s adjuvant (CFA), incomplete Freund s adjuvant (IFA), horseradish peroxidase (HRP), agarose, labelled monoclonal antibody against rabbit IgG heavy chain; Ophenylenediamine (OPD), Bovine Serum Albumin 447

3 (BSA), Coomassie blue R.250, and Tween 20 were purchased from Sigma (St. Louis, USA). All other chemicals and reagents used were of analytical grade. 2. Methods 2.1. Rabbits Immunization New Zealand white male rabbits weighing kg were acclimatized to room temperature for 2 weeks prior to immunization. Preimmune sera were obtained during this time period. Lyophilized crude venoms (50 µg/ml in physiological saline solution) were filtered through 0.22 µm sterile filter. 0.5 ml (25 µg) of venom was emulsified with equal volume of Complete Freund's Adjuvant (CFA) and injected subcutaneously into multiple sites along the back of New Zealand white male rabbits individually. Each rabbit was injected with venom of one snake species. The booster doses were made in Icomplete Freund's Adjuvant (IFA) at 4 week intervals with the doses of 50 µg up to 70 µg of the same venom. Blood from rabbits were collected after 7 days of each booster injection. The presence of antibodies in serum was determined through immunodiffusion (Ouchterlony test) and by indirect ELISA with the respective venom used as coating antigen. Finally, the immunized blood was collected and serum was stored in 4 C for bioassay tests Immunodiffusion (Ouchterlony test) Immunodiffusion experiment was carried out according to the method described by Ouchterlony [27] on glass slides using 1% agarose in Phosphate Buffered Saline (PBS 0,1M,pH 7,4) as diluent. Sodium azide in a concentration of 0.01% was added to retard bacterial growth. The titers of the antibody of each venom, were determined using serial dilution of the specific antivenom, to be tested;10µl of specific venom solutions of 2 mg/ml, was placed in the central well while in the six peripheral wells serial dilutions of the antivenom solution to be tested, were placed. After developing the precipitin bands (20-48 hours at 25 C), the plates were washed for 24 hrs in saline dried and stained with Coomassie Blue R250 for 5 min, washed with methanol acetic acid (9:1) and dried in air and photographed. The titer of each sample is defined as : the reciprocal of the highest dilution giving a positive precipitin bands with each venom [28] Purification of the crude antivenom (plasma) Antisera showing highest antibody titer (as monitored by ELISA) were collected from rabbits.this crude serum taken from hyper-immunized rabbits was subjected to antibody purification. The serum was precipitated with ammonium sulfate then cleaved with pepsin [29].The antibody molecule was split by pepsin to yield 2 identical fragments F(ab ) 2 and Fc. The Fc fragments were discarded. Each antibody was purified separately to avoid crosscontamination Protein determination Protein concentration was quantified by Lowry and al method [30], using Bovine Serum Albumin (BSA) as a standard Electrophoretic analysis of the purity of antivenoms All samples were analyzed by Sodium Dodecyl sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) at 15% under non reducing conditions according to the Laemmli method [31]. After migration, proteins were stained with Coomassie blue R Indirect ELISA for determination of antibodies titer The indirect ELISA was performed according to Theakston et al.[32] with minor modifications. In brief, Maxisorp microtitration plates (Nunc, Denmark), were coated overnight at 4 C with 100 µl of 5 µg solution of either venom in coating buffer (0,1 M carbonate buffer ph 9.6). The plates were washed three times with the PBS-T buffer (100 mm PBS ph 7.5 containing 0.05% Tween 20) in an automatic Mindray microplate washer ( MW-12A), and incubated for 1 h at 37 C with 200 µl /well of the blocking buffer (PBS containing 5% skimmed milk). Dilutions of the test Abs in the PBS-T were dispensed into triplicate wells coated with the different venoms and incubated for 1 h at 37 C. After washing, goat anti-rabbit IgG-peroxidase conjugate diluted in PBS was added (100 ml/well) and incubated for 1 h at 37 C. The plates were washed thoroughly for 3 5 times with PBS-T buffer before allowing them to react with 100 µl/well of substrate medium (potassium phosphate 10 mm, ph 7,3 containing 1mg/mL OPD and 0,06% H 2 O 2 ). The reaction was allowed to proceed for 15 min at room temperature in the dark before the addition of 50 µl /well of 2 N sulfuric acid. Absorbance was measured at 492 nm in a Mindray Microplate Reader (MR- A96). 448

4 2.7. Serum neutralization Neutralization potency of the test sera was determined in vivo using mice according to the general lines of WHO [3]. Mixtures containing challenge dose of C.cerastes and M.mauretanica venoms (5 LD 50 ), LD 50 were determined previously (data not shown), and the test antivenom (v/v) was incubated for 30 min at 37 C, inoculated individually by the intraperitoneally route (500 µl /mice) into six mice at each antivenom dilution and the survivals were recorded after 48 h. Controls which include PBS alone, venom and antivenom were included separately. The ED 50 defined as the venom concentration at which 50% of the injected mice survive and the antivenom potency expressed as (LD 50 per ml). Results were analyzed by the Spearman-Karber method [33, 35]. Antivenom potency was defined by the formula Potency = [(n -1)/ED 50 ] LD 50, where n-1 represents the number of lethal doses of the challenge minus one (theoretically the dose of venom that killed half the mice). As the ED 50 is expressed in ml and the LD 50 in mg, the final result is µg/µl (or mg/ml), indicating the milligrams of venom neutralized by 1 ml of antivenom. Results were also expressed as number of LD 50 neutralized per ml of antivenom, again discounting one lethal dose, such that LD 50 /ml= (4/ED 50 ) 1000 [35] Hemorrhagic activity It was determined by the method of Kondo et al. [36] modified by Gutiérrez et al. [23]. In the previously shaved skin of the back of swiss mice, received an intradermal (i.d.) injection of 100 µl of PBS containing increasing doses (from 2,5 to 100 µg) of each venom (C.cerastes and M.mauretanica). Mice were euthanized 2 h after injection and the areas of hemorrhage measured on the internal surface of the dissected skin. The minimum hemorrhagic dose (MHD) was defined as the amount of venom which induced a hemorrhagic area of 10 mm diameter Neutralization of hemorrhagic activity Swiss mice (n = 3 per dose level) were injected intradermally in the back with a mixture containing two minimal hemorrhagic doses (MHD) of the different venoms or pools in 0.15 M NaCl preincubated with different amounts of the antivenom (range: 5 90 µl) for 30 min at 37 C in a final volume of 100 µl). 24 h later, the mice were sacrificed, the skin excised and the major perpendicular diameters of the hemorrhagic haloes were measured at the dermal face was determined as the H-ED 50 : the dose of antivenom that decreased the hemorrhagic areas by 50% when compared with the positive controls (mice injected intradermally in the back with 3MHD of each venom or pool in the same volume). The results were analyzed by non-linear regression. Hemorrhagic neutralization potency was expressed both in terms of mass of venom neutralized (mg/ml= [3 MHD/H-ED 50 ]) to incorporate the varying doses of venom used, and in terms of the number of hemorrhagic doses (MHD/mL= [3/H-ED 50 ] 1000) at the H- ED 50 (i.e., no dose was subtracted as for neutralization of lethality) Statistical analyses Results were expressed as mean ± SD from triplicates, or with the 95% confidence intervals (C.I.) in parentheses. Student s t-test was used for comparisons and the significance of the differences between the mean values of two experimental groups was determined by the Student s t-test. Differences were considered statistically significant at values of p < RESULTS AND DISCUSSION 1. Determination of antivenom titer by immunodifusion Immunodiffusion test (Fig.1.) demonstrated the presence of several antibodies in the immunized sera of rabbits. Multiple precipitin bands were observed in C.cerastes and M.mauretanica antiserum. There was no change in the titer of antivenom solution obtained from the rabbits immunized with C.cerastes venom (Fig.1a), as compared to that of antivenom solution obtained from rabbits immunized with M.mauretanica (Fig.1b). Using the immunodiffusion experiment definite precipitin, bands were obtained with a venom concentration of 5 mg/ml, and serum dilution up to 1:32. Ouchterlony of each venom vs. produced antivenins resulted in the formation of precipitation lines between wells containing C.cerastes (Fig. 1c) and M.mauretanica (Fig.1d) venom, showed the presence of visible lines which were identical and were joined smoothly at the corners, indicating that, they had the same antigenic determinant. Significant precipitation lines developed against the antisera to C.cerastes and B.arietans snake, while weak reactions were seen against M.meuretanica, and no reaction developed against Moroccan cobra. 449

5 Noreddine Ghalim et al J. Chem. Pharm. Res., 2014, 6(12): Figure 1: a and b Immunodiffusion patterns showing determination of titer of antibody using double immunodiffusion reaction a: V C.c :C.cerastes venom (in the central well). In peripheral wells, rabbit serum antivenom raised against C.c venom, serially diluted up to 1:32. b: V M.m :M.mauretanica venom (in the central well). In peripheral wells, rabbit serum antivenom raised against M.m venom, serially diluted up to 1:32. c and d: Double diffusion reaction of rabbit serum antivenom from C. cerastes and M.mauretanica (central well) with M.mauretanica and B.a: B.arietans venoms ( c and d respectively). 2. Determination of antivenom purity by SDS-PAGE SDS-PAGE of venom revealed the presence of visible bands with molecular weights ranging from approximately 100 kda matched with F(ab )2 weight as well as the absence of albumin (Fig.2). Figure 2 : SDS-PAGE in 15 % acrylamide / bisacrylamide in non reducing conditions Lane 1: Molecular Weight Markers (MWM), Lane 2: Bovine Serum Albumin (BSA), Lane 3: F(ab )2 from commercial antivenom, Lane 4: C.cerastes F (ab') 2, Lane 4: M.mauretanica F (ab )2 3. Determination of antivenom titer by Indirect ELISA ELISA test was used to assess the antigenic titer (Fig.3) and the cross reactivity of C.cerastes and M.mauretanica antivenoms with viper and Moroccan cobra venom antigens (Fig. 4). Monospecific antivenoms from immunized rabbits with C.cerastes and M.mauretanica specific venoms were analyzed in order to evaluate levels of F(ab )2 (Fig.3). Antivenom raised against C.cerastes venom exhibited high level of F(ab )2 (1: ) compared to the M.mauretanica antivenom (1:128000). 450

6 Figure 3: Rabbit antivenom (Fab 2) titers against the venoms of C.cerastes, M.mauretanica as determined by indirect ELISA. Solutions of each venom were placed in microplates, followed by the addition of different dilutions of each antivenom and then by an anti-rabbit IgG conjugated with peroxidase. Absorbances at 490 nm were recorded, as described in the Materials and methods section. Results are presented as mean± S.D (n=3) Figure 4 : Cross- reactivity of rabbit C.cerastes ( ) and M.mauretanica () antivenom with Naja haje legionis ( ) venom (5µg/mL) (a) and with B.arietans venom (b) 451

7 4. Cross-reactivity of rabbit C.cerastes (C.c) and M.mauretanica antivenoms with Naja haje legionis and with Bitis arietans venoms Our observations demonstrate an extensive cross neutralization of C.cerastes and M.mauretanica venoms with those of B.arietans (Fig.4.A) and N.haje legionis (Fig.4.B). This result indicates that the rabbit antivenom exhibited a low level of cross reactivity with N.haje legionis venom in comparison with viper antigens (Fig.4). 5. Neutralization of lethality Neutralization is expressed as mg of venom neutralized per ml of antivenom or by the number of LD 50 neutralized per ml or vial of antivenom [16]. As shown in table 1, antivenom neutralized lethality of the venoms was tested with variable potency. In terms of median effective dose (ED 50 ), C.cerastes was neutralized most efficiently (80.4 µl of venom per ml of antivenom) whereas the M. maureantanica required the highest doses of antivenom µl (p < 0.05), with the potency values of LD 50 / ml (1.74 mg/ml) and LD 50 / ml (1.46 mg/ml), respectively. In terms of LD 50 /ml, the potency of both antivenoms showed slightly similar values (p > 0.05) (Table1). Table 1: Summary of experiments of lethality and its neutralization. LD 50, median lethal dose; ED 50, median effective dose and its conversion to potency in mg venom per ml of antivenom and to potency in LD50 neutralized per ml of antivenom Venoms LD 50 (µg) Neutralization of lethality ED 50 µl Potency mg/ml Potency (LD 50/mL) C. cerastes ± ( ) 1.74 ( ) ( ) M.mauretanica 37.04± ( ) 1.46 ( ) ( ) Results are presented as mean ± SD for a triplicate of determinations. Confidence intervals at 95% are given in brackets. 6. Neutralization of hemorrhagic activity Table 2 below shows the neutralization of hemorrhagic activity of C.cerastes and M.mauretanica by a monovalent antivenoms raised against each species. Neutralization of experimental hemorrhage, a conspicuous activity of viper venoms [34, 24], did not correlate to neutralization of lethality. In terms of mass, both antivenoms shows similar potency of neutralization of Hemorrhagic activity of C.cerastes and M.mauretanica venoms (1.47 and 1.43 mg/ml of antivenom) respectively, (p > 0.05). In terms of MHD/mL, the C.cerastes antivenom shows a highest potency (1.5-fold) than M. mauretanica antivenom (69.77 and MHD/mL, respectively) (Table 2). The results also demonstrated that monospecific antivenoms were able to effectively neutralize the hemorrhagic activity of both venoms examined. Table 2: Summary of determinations of hemorrhage neutralization. MHD, minimal hemorrhagic dose. Neutralization measurements: H- ED50, median effective dose for neutralization of hemorrhage and its conversion to potency in mg/ml and number of MHD neutralized per ml of antivenom Venoms MHD (µg) Neutralization of hemorrhage H-ED 50 (µl) Potency (mg/ml) Potency (MHD/mL) C. cerastes 21 ± ( ) 1.47 ( ) ( ) M.mauretanica ± ( ) 1.43 ( ) ( ) Results are presented as mean ± SD for a triplicate of determinations. Confidence intervals at 95% are given in brackets. In term of this study, viper bites are a serious medical problem in Morocco, particularly those occurred by C.cerastes and M.mauretanica species. The treatment of venomous snakebites can prove to be difficult due to several problems, the most conspicuous of which, is the shortage or lack of antivenoms for human use. Because commercial pressures on the pharmaceutical industry have led to a reduction in the production of antivenom in several parts of the world, their availability is sometimes rather limited and these products can sometimes hardly available and therefore impossible to obtain. Depending on the number of snake species, whose venoms are used to immunize the animals, antivenoms are either monospecific or polyspecific. Monospecific antivenoms are prepared through immunizing animals with the venom of a single species, whereas polyspecific antivenoms are prepared by using the venoms of two or more species [16, 21]. The method of double salt precipitation, thermocoagulation and enzymatic digestion (Pope method) for these fragments, is widely accepted as one of the best choices for the preparation of antivenoms for human use [10]. One of the main advantages of Pope method is the absence of regions of the immunoglobulin heavy chain. Enzymatic digestion reduces the supply of heterologous proteins to the immune system of people treated with these fragments 452

8 and, consequently, the possibility of adverse reactions by non-related to complement system activation or white blood cells, located in the portion digested by pepsin domains [37-39, 10]. These fragments are highly conserved and humoral responses against them can result in an Arthus reaction in the subsequent treatment with antivenom [40]. Furthermore, these preparations are safe with respect to the occurrence of immediate adverse reactions, as complement activation would be only slightly greater than that observed using F (ab') 2 [41, 42]. Hence, to increase the purity of their products, antivenom manufacturers should expend greater in order to improve the plasma fractionation process and subsequently generate formulations free of non-immunoglobulin protein impurities [43-45]. The results obtained in the present work show that it is possible to produce quality antivenoms with a considerable reduction of purification steps and production costs. This very fact, leads us to assume that the production of inexpensive antivenoms and good quality products for human use is possible and should therefore be fostered and encouraged. The key to this type of production is to obtain high titers of antibodies with a low production cost. In this work, we immunized rabbits with venom of Moroccan vipers to obtain hyperimmune plasma. The plasma was processed to separate whole IgG of F(ab ) 2 fragments using the double saline precipitation and pepsin digestion conventional methods. The obtained antivenins were tested for their biochemical and immunochemical characteristics and neutralizing potency. The electrophoresis pattern of each antivenom (Av-C.cerastes and M.mauretanica ) performed showed only one protein fraction matched with band in the order of 100 kda corresponding to F(ab ) 2 fragments. The presence of albumin or contaminants of high or low molecular weight was not detected in any of the preparations (Fig.2). The present study describes the active immunization of rabbits by C.cerastes and M. mauretanica venom joined smoothly at the corners indicating that there was no change in the antigenic determinants. The results obtained in the present work are in accordance with those of other investigators. There was no change in the titer of antivenin solution obtained from the rabbits immunized with the Cc venom, as compared with that of antivenin solution obtained from rabbits immunized with M.m venom, using the immunodiffusion venom concentration of 2 mg/ml, and serum dilution up to 1:32. Both vipers were immunogenic and induced specific antibody formation. Antivenom cross reactions have been studied using immunodiffusion and ELISA. Cross precipitation does not necessarily mean that there is cross protection. However, a cross protection is generally observed between closely related species. A very important cross neutralization exists between venoms of Moroccan vipers, as it is shown by Ouchterlony test (Fig.1), monovalent C.cerastes and M.mauretanica antivenom neutralizes different Moroccan vipers venom (Cc, Mm and Ba), but not that of Naja haje (Fig.1), which is characterized by the presence of a neurotoxin, crotoxin [46] which is lacking in C.cerastes and M.mauretanica [47, 48]. However, there is a cross reaction observed with these two antivenins against B.arietans and N.haje legionis venom, in spite of the fact that it was not used in the immunization procedure (Fig. 4.B).These findings agree with previous investigations, that confirm the high level sensitivity of ELISA method [50]. The 50% protective doses (ED 50 ) of the antivenoms were given according to the in vivo method, which consists of inoculating several batches of mice with a mixture containing 5 DL 50 of venom and increasing volumes of antivenom incubated for 30 min at 37 C. The two antivenoms proved to be effective against the Cc and Mm venom. Both antivenoms obtained in the immunization process were efficient in neutralizing the venoms of the snakes studied. Considering the neutralizing of hemorrhagic activity obtained by both antivenoms, our results have shown that no important differences were observed in the neutralizing potency of the antivenins; the differences in the neutralizing potency, were not statistically significant (p > 0.05). These results have also demonstrated the high efficiency of both the monovalent C.Cerastes and M.mauretanica antivenoms in the treatment of bites by C.Cerastes as well as M. meuretanica envenomation. The data emphasizes the potential value of having monovalent C.cerastes antivenin and M.mauretanica, especially in localities where both snakes are prevalent and where the identification of the offending snake is difficult or impossible. CONCLUSION In conclusion, purity is an important issue that must be determined to evaluate the quality of biological products. In the case of antivenoms, the evaluation of purity can be performed by two different approaches, i.e. physicochemical 453

9 analysis and immunochemical analysis. The Electrophoretic analysis, which is currently used to determine the absence of non-immunoglobulin contaminant proteins (i.e. albumin or non-antibody globulins), should be supplemented by the assessment of the potency and ELISA titer,especially that the latter ensures the reduction of the use of mice. Such immunochemical assessment, with electrophoretic analyses, constitutes a more complete estimation of antivenom purity. Precipitation and pepsin digestion are steps to be considered in the production in order to enhance the antivenom efficacy. Results of cross-reactivity studies were in line with the phylogenic relationship between C. cerastes, M.mauretanica and B.arietans, and supported cross-neutralization. Monospecific antivenoms raised against venoms of C. cerastes, and M. mauretanica, were found to be effective in neutralizing the lethality of venoms of C.cerastes and M.mauretanica. These results suggest that the monovalent antivenom may be used as a treatment against viper envenomation, especially by B.arietans. C.cerastes and M.mauretanica on the other hand, induce lethal, hemorrhagic activities in mice. Monospecific antivenom raised against each species was highly effective in the neutralization of toxic and hemorrhagic activity of the venom tested. It should finally be noted that physicochemical or immunochemical techniques alone offer only partial information of antivenom purity and, as a result of this both types of analysis are required to carry out an integrated assessment of this parameter. Besides this, we propose that an increase of snake antivenom titer can be achieved by the modification of immunization schedules. Ethical Statement All the testing and procedures involving animals strictly followed the ethical principles in animal research adopted by the World Health Organization [34]. Acknowledgments The authors are very grateful to Pr Maliki My Sadik - Department of English Faculty of Art and Humanties- Ain Chock, Casablanca, who proofread and edited a first version of this paper. REFERENCES [1] JM Gutiérrez; D Williams; HW Fan; D A Warrell, Toxicon, 2010, 56, [2] World Health Organization. Neglected Tropical Disease: Snakebite, WHO, Geneva, 2009 [3] World Health Organization. Guidelines for the prevention and clinical management of snakebite in Africa, WHO Regional Office for Africa, Brazzaville, [4] World Health Organization. Rabies and Envenomings, A Neglected Public Health Issue, World Health Organization, Geneva, [5] F Chafiq; N Rhalem; L Ouammi; M Fekhaoui; I.Semlali; A Soulaymani; R Soulaymani, Toxicologie, 2011, 9, 6-9. [6] H Lallie ; H Hami ; A Soulaymani ; F Chafiq ; A Mokhtari ; R Soulaymani, Med. Trop., 2011, 71, [7] H Lallie; H Hami; A Soulaymani; F Chafiq; M Fekhaoui; A Mokhtari; M Esmaili ; R Soulaymani, Bull. Soc. Pathol.Exot., 2012, 105, [8] A Arfaoui ; R Hmimou; L Ouammi; A Soulaymani; A Mokhtari; F Chafiq; R Soulaymani- Bencheikh, J. Venom. Anim. Toxins. Incl. Trop. Dis., 2009, 15, [9] Calmette A., L'immunisation artificielle des animaux contre le venin des serpents, et la thérapeutique expérimentale des morsures venimeuses, C. R. Soc. Biol., 1894, 46, [10] J P Chippaux; M Goyffon, Toxicon, 1998, 36 (6), [11] J M Gutiérrez; RDG Theakston; DA Warrell, PLoS. Med., 2006, 3,150. [12] BK Nelson, Med. Toxicol., 1989, 4, [13] J B Jr Sullivan, Ann Emerg Med, 1987, 16, [14] S K Sutherland, Med. J.Aust., 1992, 157, [15] D A Warrel. Guidelines for the Management of Snake-bites, Organisation Mondiale de la Santé, 2010, 152. [16] World Health Organization. Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins, WHO, Geneva,2010. [17] S K Sutherland. Med. J. Aust., 1977, 1, [18] G León; M A Rodríguez; A Rucavado; B Lomonte; J M. Gutiérrez, Biologicals, 2007, 35 (1), [19] F LoVecchio; J Klemens; EB Roundy; A Klemens. Wilderness Environ. Med., 2003, 14,

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