The Verification of the Efficacity of the Polyspecific Antisorpionic Serum against Three Scorpion in El Oued Region

 

Souhaila Meneceur¹, Mohammed Ridha Ouahrani¹, Salah Eddine Laouini²,

Rachida Zouari Ahmed¹, Abderrhmane Bouafia^2*

¹Department of Chemistry, Faculty of Exact Sciences, University of Echahid Hamma Lakhdar,

El-Oued, 39000, Algeria.

²Department of Process Engineering, Faculty of Technology, University of Echahid Hamma Lakhdar,

El Oued 39000, Algeria.

 

ABSTRACT:

In Algeria and like many other countries of the world and in the absence of other effective therapeutic remedies. Antidepressant serum remains the only way to neutralize these toxins and fight these disorders despite sometimes-unsatisfactory results. In order to contribute subsidize to the improvement of these results; we considered it useful to study the factors influencing the composition and the concentration of the toxins to know the behavior of the antiscorpionic serum on these toxins. To do this, we tested the ability of polyspecific antisorpional serum and determined whether enough antivenom was administered. Comparative titrations were performed separately on the venoms of female scorpions (Androctonus australis hoctor, Buthacus arenicola and Androctonus Amoreuxi) caught in three different geographical region in El Oued: Debila, Reguiba and Hassi Khalifa.

 

 

 

INTRODUCTION:

Scorpion envenomation is a grave medical problem and a real danger in a number of countries around the world, mostly in central and southern America, northern Africa, the Middle East and India^1-5. This specific pathology represents one of the most significant public health difficulties in Algeria and more particularly in the regions of the Highlands and the South for example the region of Oued Souf where, every year, several thousand people are stung by the scorpions, of which about ten die on average.

 

Although anti-scorpionic serum is the only remedy against scorpion envenomation, its administration is currently much questioned and this, given its ineffectiveness in many cases. For an effective contribution to the improvement of the therapeutic management of people envenoms, and to decrease the rate of lethality, it is clear to develop studies of the venoms in their compositions and modes of action because they are the resource essential manufacturing of SAS^6-8.

 

Antivenom remains to date the only definitive behavior for envenomation^9-12. The sustainability of antivenom source has been excessive contest worldwide with financial restraint being mentioned as the major causes especially in developing countries¹³. Then, the current therapeutic procedures for antivenomic must be optimized for the pharmacokinetics of venoms/toxins and how this serum might modify the clinical pharmacokinetic profile of venoms/toxins should be promoted, which is essential for better adaptation serum dosages of antivenom. Various methods of treating scorpion fever are treated, ranging from symptomatic treatment to antivenom serum. Advocates of the latter refer to the use of inappropriate antioxidants and doses^14-19. On the other hand numerous clinicians have questioned the effectiveness of antivenom therapy^20-24. Thus, this study evaluated the efficacy of antivenom produced in Algeria three different venom obtained three scorpions species.

 

RESULTS AND DISCUSSION:

Analysis of the toxic fraction:

Fig.1 shows a map corresponding to three areas where the ninety scorpions were collected for the treated venom as described in the materials and methods section, after which the samples were subjected to chromatographic analysis. 

Median lethal dose (LD50):

Scorpion venom comprises a small structure of neurotoxin polypeptides containing simple, low-molecular-mass proteins that have lethal and paralytic effects. Venom toxicity differs allowing to numerous factors such as genus, species, age, physiology, feeding state and region of the scorpion. Analysis of the toxic fraction Fig.1 shows a map corresponding to three zones in which ninety scorpions were collected for the treated venom, as described in the materials and methods section, after which the samples were analyzed to chromatographic.

 

Previously, the main difficulties were related to the standardization of venom quality²⁵. To develop an antivenom serum that neutralizes as far as possible the toxic effects of venoms, we necessarily have a high class of venom with a high toxic activity (LD50). The procedure of venom collecting effect on activity²⁶. The results were described in Table 1. The LD₅₀ of the native venoms of Androctonus australis hector, Androctonus Amoreuxi and Buthacus arenicola were respectively estimated at 0.35mg/20g, 0.48mg/20g, and 0.90mg/20g. The value of median lethal dose highlighted that toxicity was high for venom obtained from Androctonus australis hector and Androctonus Amoreuxi presented moderate toxicity. The lower value founded for venom obtained from Buthacus arenicola.

 

High performance liquid chromatography (HPLC):

Fig.2 shows the HPLC spectra obtained by chromatographic separation of soluble venoms in a C18 reverse phase column. For a better transparency, the different compounds determined by this technique have been alienated in Fig.1: (A) Androctonus australis hector from the region of Debila, (B) Androctonus australis hector scorpions from the region of reguiba and (C) venom obtained from Androctonus australis hector from Hassi khalifa region. The spectra D and E presented the venom obtained from Androctonus Amoreuxi, and Buthacus arenicola respectively of Debila region. Near analysis of these venom exposes similar characteristics. The most important components are related to the major components and time of elution, but the relative concentrations appear to be variable.

 

This is already well documented by similar analyzes shown on other venoms, especially when the number of individuals used for the preparation of the pooled mixture is low^27-29. As can be seen in Fig.1, the venom obtained from different species contained a mass of bioactive components. Although HPLC was able to isolate these fractions in a commendable manner, the yield obtained was not sufficient to allow us to use a purified component for the injection of a camel.

 

Venoms include some carefully related profiles based on key components and elution timing, but relative concentrations appear to be modifiable. This was previously well documented by similar analyzes conducted on other venoms, particularly when the number of individuals used for the preparation of the shared mixture was not important^27,28.

 

Venom–antivenom complex:

The complex of venom/antivenom assay will single detect bound antivenom for the microplate is coated with an anti-scorpion venom antibody which fixes the venom, and detection is by labeled anti-horse antibodies which bind horsy antivenom (Fig.2: A, B and C). In vitro, this delivers an amount of antivenom and venom binding for increasing concentrations of antivenom. The curve increases with the increasing binding of the antivenom to the free venom pending a point where increasing amounts of antivenom stop the binding of the venom-antivenom complex to the microplate, as there are no longer any binding sites of free antibodies on venom particles. The venom/antivenom peaks are the concentration at which each venom component is regularly bound to at least one antivenom molecule. This gives us a new amount of antivenom capacity. In addition, it allows an assay to measure the venom bound in vivo and to control if the venom identified post-antivenin using the free venom assay is bound. For minimum concentrations of antivenom, the antivenom fixes to the venom molecules in a one to one fraction to form complexes.

 

Fig.1. HPLC analysis of soluble venom from different geographical areas: A 0.02 mg protein portion from each toxic fraction of venom was separated into a C18 reverse-phase column eluted with a gradient from solution A 0.12% trifluoroacetic acid (TFA) in water to 60% solution and B 0.10% TFA in acetonitrile run for 30 minutes. (A, B, C) the venom of scorpions Androctonus australis hector collected in Debila, Reguiba and Hassi khalifa. (D, E) the venom of scorpions Androctonus Amoreuxi and Buthacus arenicola collected in Debila.

 

 

The venom/antivenom complex immobile has free binding locations on the venom molecule which lets more antivenom to bind with increasing concentrations to form (venom/antivenom)_n wherever n is the maximum number of antibody binding locations on a venom molecule. In another hand, at least one group sets the free and open remains of need for the venom-venom complex to bind to the anti-venom scorpion antibodies of the microplate. This is the reason why, firstly, as the concentration of antivenom increases and the percentage of antivenom in the mixture increases, an increasing amount of venom venom is identified. Concentration of the peak complex follows after further binding of the antivenom resulted in a decrease in free antibody binding sites on the venom molecules, resulting in a decrease in microplate binding. The complex peak occurs when there is regularly or mainly in the venom/antivenom 1 mixture, which means that there is at least one antivenom molecule involved in each venom molecule.

 

This is description it possible that of what occurs for the reason that venom consists of diverse toxins and every toxin is possible to have a different number of antibody binding places reliant on toxin size and antigenicity. Furthermore, antivenom is a polyspecific antibody to different toxins and different toxin epitopes with varying affinities. However, the stepwise formation of (venom/antivenom)k complexes (where 1 < k for Fig.2 A and B, and k=0 for figure C) relates to the behavior of the whole population of venom (toxins) and antivenom molecules, anyway of the fact the venoms comprise dozens of different proteins, each with some epitopes, and that the antivenom are themselves polyspecific. For most antivenom-venom couples, the variety of similarities for the first and second linkage sites is not great, so the venom / antivenom peak would not be so distinct in most cases. We therefore consider an average set of antivenom-venom pairs for a range of n and k. The remark that the venom / venom curves of separate components of the venom do not differ too much from those of the whole venom justifies this opinion (Fig.2, A and B). In certain cases, a different antivenom-venom curve was not attained (Fig.2 C). However, the curves do return on the way to zero, showing that the venom can be completely neutralized by the antivenom; with scorpion antivenom provided a wide peak, possibly suggesting a low similarity of this venom for scorpion antivenom.

 

The data were adjusted to the difference of two exponential curves empirically to allow calculation of the maximum absorption point by interpolation. In the measure of the venom / antivenom value, the strong indication of the antivenom / venom mix is regular, which means that each molecule of venom is involved in the smallest molecule of antivenom. The efficacy marker can be applied because it means that all the venom molecules (toxins) are linked to at least one antibody, so that they can not be distributed at their place of action and / or can be eliminated. . This venom-to-venom ratio is restricted over a range of venom concentrations because the slope appears constant (Fig.3). The results are shown in Table 1; remarkably, these values between 0.038 and 0.018 U are necessary for 1 μg of venom. While "neutralizing" is not defined, it can be said that the fixation of at least one molecule of antivenom to a molecule of venom, even if it is not close to the active position of the molecule, is sufficient to prevent it from getting out of the circulation and solidify it to elimination by the reticuloendothelial system or circulating phagocytes.

 

 

Fig.2. Logarithmic-linear plots of venom–antivenom (VAV) measurements versus antivenom concentration for mixtures of venom and antivenom, at three or concentrations of venom (ng/ml) – A: Androctonus australis hector, B: Androctonus Amoreuxi, C: Buthacus arenicola each collected in Debila. The plotted points are the average of three absorbances. For panels A and B the line is a predicted curve based on the subtraction of two exponential curves. For panel C a curve could not be fitted and the lines connect experimental points.

Fig.3. The plot of the antivenom concentration at which the maximum venom/ antivenom absorbance occurred against the concentration of venom for two species of scorpions venoms (Androctonus australis hector and Androctonus Amoreuxi collected in Debila).

 

Table 1: The ratio of antivenom concentration to the concentration of venom at the maximum venom/antivenom absorbance for Algerian commercial antivenom (SAS.)

MATERIALS AND METHODS:

Venom:

Scorpions of three different species (Aah, Aam and Buthacus arenicola) were collected in the Oued Souf region of Algeria. The venom of AAH was obtained by electrical stimulation of ninety female scorpions from different geographical areas as follows³⁰: sixty from Debila, fifteen from Hassi Khelifa and the same number from Reguiba. The crude venom was dissolved in sterile double-distilled water and centrifuged at 13,000 x g for 10 min at 4°C. The precipitate was discarded and the supernatant kept at 20°C until needed. After a captive day, individual scorpions from each area and age were treated and pooled with venom excretion collected in small ampoules. The venom is stored at -20°C³¹.

 

Antivenin:

The universal anti-scorpion antivenom was kindly given by public hospital center of Debila, and prepared by the Institute Pasteur of Algeria in 5ml bottles (lot n° 40313/4 Manufactured: May 2013 Expiration: Apr 2017). This preparation was obtained from the serum of horses hyperimmunized with from the venom scorpion of Androctonus australis Hector. Algerian commercial antivenom was produced by Institute Pasteur of Algeria, including polyspecific scorpion, each 1ml of serum contains immunoglobulins F(ab ')2 neutralized minimum 1500 toxic units of Aah dry venom. The commercial antivenom is of equine origin.

 

Determination of the median lethal dose (LD50):

The lethal potency of venoms, as measured by LD₅₀ significance resolve (in micrograms of dried venom per mouse), was evaluated as suggested by the World Health Organization. Meetings of for mice (22-20 g) were used per dose of venom. The venom doses were attuned with NaCl 150mM and final was injected in volumes of 500 μl through intravenous routes³². The rate mortality was verified 24 h after injection. The LD₅₀ is calculated using the arithmetical method of Karber^33,34, which is as follow:

 

Where, LD₅₀ = Median lethal dose

LD₁₀₀ = Least dose required to kill 100%

a = Dose difference

b = Mean mortality

n = Group population.

High performance liquid chromatography analysis (HPLC)

 

The soluble venom from each locality and species was applied to the high-performance liquid chromatographic (HPLC) system. A high performance liquid chromatography system, Shimadzu LC 20 AL equipped with a universal injector (Hamilton 25µL) SPD 20A, UV-VIS detector SPD 20A (Shimadzu) was used.

 

Typically, 0.02mg dissolved in 2ml was applied each time. An analytical C18 reverse-phase column (dimensions of 250 × 10mm). Components were purified using a linear gradient from 0% solution. A [0.1% acetic acid in water] to 60% solution B (0.10% acetic acid in acetonitrile), run for 35 minutes²⁹. The detection was monitored by absorbance at 230nm with 0.5-unit sensitivity and eluted at 1ml/min.

 

Venom–antivenom complex (VAV) measurement:

Bovine serum albumin (BSA) and tetramethylbenzidine (TMB) were all purchased from Sigma. All reagents used were of analytical grade. Carbonate buffer is 0.1 M, pH 9.5. Blocking solution is 0.5% BSA in phosphate buffered saline (PBS) at pH 7.4. The above mixture was washed with a solution 0.02% Tween 20 in PBS. The microplates were used and the absorbance was calculated at 492nm (RI 2100C microplate Reader plate reader). All procedures were carried out at room temperature.

The different known concentration of venom in obstructive solution was added to sequential dilutions of antivenom in PBS (1/1000), such that the final venom concentration in the mixture was 100, 300, 600ng/ml. The mixture was permitted to stand for one hour, all experiments carried out in triplicate. Control solutions containing antivenom only were included to allow for subtraction of background absorbance. Microplates were covered with anti-scorpion venom IgG conjugated (0.1 M in carbonate buffer, pH=9.5) for 24 h at room temperature than at 4°C overnight. They were then splashed once for 5 minutes and blocking solution (100 µl) was useful for 1 h. Microplates were washed twice time for 5 minutes and an incubated mixture of venom/antivenom (100µl) was added for 1 hour. Finally, the microplates were washed three times for 5 minutes and a solution of labeled anti-horse IgG (100µl in blocking solution) was useful. After a more hour the microplates were washed 4 times, and TMB (10µl) was applied, followed by H₂SO₄ (50µl, 2 M) to stop the reaction ³⁵.

 

CONCLUSION:

In this investigation, we have only presented the detection of venom/antivenom in vitro mixtures of venom and antivenom. The antivenom presented an important role in the actions of envenomation by scorpion stings. By comparing between three venom scorpion Androctonus australis hector, Androctonus Amoreuxi and Buthacus arenicola; the venom obtained from Androctonus australis hector showing the high toxicity value. The lower toxicity value founded for venom obtained from Buthacus arenicola. The antivenom used as antigen neutralize strongly the venom of Androctonus australis hector and Androctonus Amoreuxi but presented any reaction against Buthacus arenicola.

 

CONFLICTS OF INTEREST:

The authors declare that there are no conflicts of interest.

 

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Received on 06.06.2020                    Modified on 01.07.2020

Accepted on 16.07.2020                   ©AJRC All right reserved