EVALUATION OF THE NEUTRALIZING CAPACITY OF Androctonus crassicauda ( OLIVIER , 1807 ) ANTIVENOM AGAINST Leiurus quinquestriatus

The two most venomous species of the family Buthidae, Leiurus quinquestriatus and Androctonus crassicauda, are found in Africa and in the Middle East. Potency and paraspecific activities of A. crassicauda antivenom (RSHC antiAc) were tested against L. quinquestriatus venom. The sera produced by Refik Saydam Hygiene Center (RSHC) showed strong reactivity against the venoms of A. crassicauda and L. quinquestriatus in western blotting and dot-blot analysis. RSHC anti-Ac presents immunoactivity and neutralizing potential against Leiurus quinquestriatus venom. Neutralization capacity of antivenom was found to be 400 μL against 40 minimum lethal doses (MLD) of A. crassicauda scorpion venom and 10 MLD of L. quinquestriatus venom. This study indicates that the RSHC anti-Ac could be used for treating L. quinquestriatus stings.

Apart from antivenom, there is still no specific treatment for envenomation by scorpions.However, the antivenom's role in the treatment of scorpion stings remains controversial and its effectiveness depends on its potency (4,11,39).In Turkey, scorpion antivenom has been produced at the Refik Saydam Hygiene Center (RSHC) since 1942 by immunizing horses with macerated A. crassicauda telsons (27,41,42).In addition to its efficacy against A. crassicauda envenomations, RSHC anti-Ac has been reported to be useful following the stings of other scorpion species as well (28,41,43).
Paraspecific activity of antivenom has clear practical advantages (if species-specific antivenom is unavailable), particularly in remote regions.Therefore, identification of the factors that lead to effective antivenom production is crucial.In this study, the potency and paraspesfic activities of RSHC anti-Ac will be presented against L. quinquestriatus venom.

Scorpions
A. crassicauda scorpions were collected in the southeastern Anatolia region (Mardin); L. quinquestriatus scorpions were collected in northern Iraq.Animals were

Experimental Animals
Female Swiss albino mice weighing 25 ± 1 g were employed to determine minimum lethal dose (MLD), and minimum effective dose (MED) (28).They were kept in the experimental room at ambient temperature with 60 ± 10% humidity and were fed commercial rodent pellets and water ad libitum throughout the experiment.

Venom
Venom was milked from mature scorpions by telson electrical stimulation (28).It was diluted with equal volume of sterile double-distilled water and then centrifuged at 15,000 rpm for 15 minutes at 4°C.The supernatant was removed and immediately stored at -20°C until use.

Antivenom
Antivenom was prepared from the same pool of hyperimmune serum obtained from horses immunized against A. crassicauda venom, according to an immunization protocol described by Ozkan et al. (27).Increasing venom doses, mixed half-and-half with adjuvants, were injected subcutaneously into horses on the 1 st , 14 th , 21 st , 28 th , 35 th and 42 nd days.On the 45 th , 48 th and 51 st days, blood samples were collected three times from the jugular vein of each animal and stored in containers with 10% sodium citrate.After plasma separation, antivenom was obtained, from combined plasma, by the digestive method and kept in the dark at 4°C (27).One dose of RSHC anti-Ac was normalized to neutralize 2 MLD of A. crassicauda venom in rats when tested subcutaneously (1,27).

Lethality Assay
The MLD for A. crassicauda and L. quinquestriatus toxins was determined by subcutaneous (SC) administration of venom, diluted in physiological saline solution (PSS) [0.9% (w/v) NaCl], to mice as described previously (28).Groups of five mice received A. crassicauda and L. quinquestriatus venoms at doses ranging from 1 to 12.5 µL/mouse (for L. quinquestriatus) and 2.5 to 15.0 µL/mouse (for A. crasssicauda).The injected venom volume was kept constant at 500 µL/mouse.The control group received PSS.Following treatment, the animals were monitored for 48 hours and lethality was recorded at the end of the experiment (28).The minimum dose that kills 100% of animals is considered the MLD (Table 1).

Serum-neutralization Assays
To a series of tubes containing equal amounts of each individual venom, four different quantities of A. crasssicauda antivenom were added.A 2.5 mL solution containing 40 MLD from A. crasssicauda and 10 MLD from L. quinquestriatus venom were prepared in PSS, mixed with the antivenom and incubated at 37°C for 60 minutes.Groups of six mice were injected SC with these mixtures (Table 1).The injection volumes were kept constant at 0.5 mL/mouse in all groups.The control group was injected with PSS-venom solutions.The number of surviving mice was recorded for 48 hours after injections and the antivenom dose that protected mice was considered efficacious.The effective dose of horse serum was expressed as MED (28).

SDS-PAGE, Western Blotting and Dot Blot Analysis
Venoms were initially analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (23).Venom samples of A. For dot blot analysis NC membranes were divided into 1 cm 2 sections and samples of diluted venom (2 µL, 3 µL and 4 µL) were applied to the center of the grid by using a narrow-mouth pipette tip.The membranes were dried for 180 minutes at room temperature and a procedure similar to western blotting was followed from this point onward.Briefly, the membranes were blocked for one hour using BSA and later incubated with the RSHC anti-Ac (1:1000) for one hour.The membranes were then washed and incubated with HRP-conjugated anti-horse antibody (1:5000) for 60 minutes.Afterwards, they were rewashed before reacting with Immun-Star® HRP Chemiluminescent substrate.Finally, the membranes were exposed to an X-ray film and developed in a darkroom.

RESULTS
The minimum lethal doses of the scorpion venoms were found to be 5 and 10 µL by SC injection, respectively, for L. quinquestriatus and A. crassicauda (Table 1).The potency of RSHC anti-Ac (500 µL) has previously been determined to neutralize 2 MLD in 150 g rats according to instructions.Neutralization capacity of RSHC anti-Ac was observed to be 400 µL against 40 MLD of A. crassicauda venom and 10 MLD of L. quinquestriatus venom, while all mice died in the control groups.
The antivenom reacted strongly with A. crassicauda venom.Western blotting also showed the presence of L. quinquestriatus venom components recognized by RSHC anti-Ac.Western blotting indicated proteins between 39,000 and 191,000 Da in both venoms (Figure 1).
Different protein concentrations in venoms were also estimated by dot blot.Figure 2 shows the strong reaction of L. quinquestriatus venom protein with RSHC anti-Ac.
High-performance liquid chromatography (HPLC) was employed to obtain the chromatographic profile of A. crassicauda venom, from which about 25 compounds were discretely separated as shown in Figure 3 (unpublished data).
Venom analysis by mass spectrometry (LC-ESI-HRMS) of [MH]+ detected 42 major components within 437 to 44.737 Da, as shown in Table 2 (unpublished data).
crasssicauda and L. quinquestriatus were separated on precast NuPAGE® 12% Bis-Tris gels (Invitrogen Corporation, USA) and were electrophoretically transferred to a nitrocellulose membrane (NCM) and divided into two sections.The membranes were incubated in blocking buffer -3% bovine serum albumin (BSA) in Tris-Buffered Saline Tween-20 (TBST -0.1% Tween 20, 150 mM NaCl, 10 mM Tris-Cl, pH 7.4) (Sigma-Aldrich, USA) -for 60 minutes.The membranes were washed three times with TBST and then their strips were exposed to RSHC anti-Ac (1:4000 concentration) for one hour.Membranes were again washed three times with TBST and subsequently incubated with horseradish peroxidase (HRP) conjugated anti-horse antibody (1:5000) for 60 minutes.The membranes were washed with TBST for 10 minutes and antigens were visualized using Immun-Star® HRP Chemiluminescent substrate (BioRad, USA).Membranes were exposed to an X-ray film in a darkroom and developed.

Figure 3 .
Figure 3. Mass spectrum of total ion chromatogram and UV trace of the crude venom.Total ion chromatography of A. crassicauda venom between 6 and 49 minutes, with peak identifications.

Table 1 .
The MLD was determined for L. quinquestriatus and A. crassicauda venoms by SC injection.Neutralization capacity of antivenom was assayed for both venoms *MLD: