J Arthropod-Borne Dis, December 2015, 9(2): 176–183 SA V Zoelen et al.: Antigenic Cross-Reactivity …

Original Article Antigenic Cross-Reactivity Anti-Birtoxin against Androctonus crassicauda

*Suhandan Adigüzel Van Zoelen 1, Ozcan Ozkan 2, Bora Inceoglu 3

1Netherlands Organisation for Applied Scientific Research-Innovation for life-Hollanda, Turkey 2Drug and Medical Device Agency of Turkey, Ankara, Turkey 3Department of Entomology, University of California, Davis, USA

(Received 2 Nov 2013; accepted 7 May 2014)

Abstract Background: Antivenom is still widely used in the treatment of envenomation as there are no vaccines or other effective agents available against animal . Recently, named birtoxin family have been described from Parabuthus transvaalicus and Androctonus crassicauda. The aim of the present study was to test the anti- birtoxin for their ability to neutralize the lethal effects of A. crassicauda venom. Methods: SDS-PAGE and Western blotting used the presence of components from A. crassicauda and P. transvaalicus scorpion venoms and to determine the degree of cross-reactivity. The Minimum Lethal Dose (MLD) of venom was assessed by subcutaneously (sc) injections in mice. Results: The MLD of the A. crassicauda venom was 35 µg/ 20g mouse by sc injection route. Western blotting showed the presence of components from A. crassicauda and P. transvaalicus scorpion venoms strongly cross react with the A. crassicauda antivenom. However, Western blotting of the A. crassicauda scorpion venom using the Refik Saydam Public Health Agency (RSPHA) generated antibody showed that not all the venom components cross reacted with the anti-birtoxin antibody. The antibodies only cross reacted with components falling under the 19 kDa protein size of A. crassicauda venom. Conclusion: The bioassays and Western blotting of A. crassicauda venom with the anti-birtoxin antibodies produced against a synthetic peptide showed that these antibodies cross reacted but did not neutralize the venom of A. crassicauda.

Keywords: Androctonus crassicauda, Venom, anti-birtoxin antibody, Cross-reactivity

Introduction

Most of the medically important scorpion that 100.000 distinct peptides exist in scor- species belong to Buthus, Parabuthus, pion venom but only limited number of these Mesobuthus, Tityus, Leiurus, Androctonus peptides have been described (Possani et al. and Centruroides genera of the Buthidae 1999, 2000, Martin-Eauclaire et al. 2005, family (Balozet 1971, Bücherl 1971, Efrati Inceoglu et al. 2006). 1978). Scorpion venoms can be classified The unique specific treatment of scorpion into two groups according to their molecular envenomations is immunotherapy with anti- sizes, long-chain and short-chain neurotox- bodies from immunized horses (Ghalim et ins. The short-chain neurotoxins are 3,000 to al. 2000). However, the venom is a complex 4,400 Da and act on potassium or chloride mixture of antigens wherein not all compo- channels. Long-chain neurotoxins are 6,500 nents are equally important for the produc- to 7,800 Da and act mostly on sodium chan- tion of neutralizing antibodies. Thus, the iden- nels (Possani et al. 1999, 2000, Inceoglu et al. tification of immunogenic protein(s) and/or 2006, Ozkan et al. 2008). It has been estimated their neutralizing epitopes may lead to the 176 *Corresponding author: Dr Suhandan Adigüzel Van http://jad.tums.ac.ir Zoelen, E-mail: [email protected] Published Online: March 11, 2015  J Arthropod-Borne Dis, December 2015, 9(2): 176–183 SA V Zoelen et al.: Antigenic Cross-Reactivity …

use of more clearly defined substances as blood samples were collected three times immunogens to develop efficient antivenoms from the jugular vein of each animal and or to their use as antigens. stored in containers with 10 % sodium The venom of P. transvaalicus consists of citrate. After plasma separation, antivenom recently described closely related neurotoxins was obtained, from combined plasma, by the named birtoxin family (Inceoglu et al. 2001, digestive method and kept in the dark at 4 2005). An antibody developed using a ºC. One dose of RSHA anti-Ac was synthethic peptide composed of the first 18 normalized to neutralize 2 MLD of A. amino acid residues of birtoxin displayed crassicauda venom in rats when tested strong reactivity with the whole venom of P. subcutaneously. transvaalicus, P. leisoma and pure birtoxin (Inceoglu et al. 2006). These antibodies also Anti-birtoxin antibody neutralized the venom of P. transvaalicus in The 18 residues N-terminal portion of mice. Recently, Calışkan et al. (2006) also birtoxin-like peptides ‘NH2-ADVPGNYPLD reported the presence of peptides in A. KDGNTYKC’ was commercially synthesized crassicauda venom that belong to the by Sigma and polyclonal antibodies against birtoxin-like peptide family. this peptide were raised by Sigma-Genosys In this study, we tested the anti-birtoxin (Inceoglu et al. 2006). Briefly, the synthetic antibodies for their ability to neutralize the peptide was cross-linked to keyhole limpet lethal effects of A. crassicauda scorpion hemocyanin and rabbits were immunized. venom. The bleedings were done after the 4th, 5th and 6th booster doses and pooled. IgG molecules Materials and Methods were purified using a Protein A antibody purification kit from Sigma following the Venoms manufacturer’s instructions. Protein concen- Venom was obtained from mature A. trations were determined using a BCA kit crassicauda (from Sanliurfa) by (Pierce, USA) with ovalbumin as the standard. electrical stimulation of the telson. The ven- om was mixed with sterile double-distilled Determination of the Minimum Lethal Dose water and centrifuged at 15,000 rpm for 15 (MLD) in mice min at 4 ºC. The supernatant was immedi- All the experiments were performed ac- ately lyophilized at Refik Saydam Public cording to the guidelines by the ethical com- Health Agency (RSPHA) and stored at -80 mittee of the Faculty of Veterinary Medicine oC until use. Venom of commercially ob- in Ankara University. The Minimum Lethal tained P. transvaalicus scorpions were col- Dose (MLD) of venom was assessed by lected as described (Inceoglu et al. 2001, subcutaneously (sc) injections in mice (20±2 2006) at University of California, Davis, CA. g). The animals were kept in the experiment room under standard conditions throughout Antivenom (RSHC anti-Ac) the experiment. Five mice per each dose- Antivenom of A. crassicauda was obtained group were injected sc with doses of venom, as described (Ozkan et al. 2006a). Briefly, diluted in 0.5 ml saline solution. An equiva- increasing venom doses, mixed half-and-half lent volume of 0.5 ml saline was injected with adjuvants, were injected subcutaneously into five mice as negative control group. The into horses on the 1st, 14th, 21st, 28th, 35th and animals were observed for 48 h after venom 42nd days. On the 45th, 48th and 51st, days, injection in order to determine MLD.

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Serum-neutralization assays in mice Gel electrophoresis of the venoms and West- A solution of A. crassicauda venom (3 ern Blotting MLD for each mouse) diluted in physiologic Venoms were analyzed by sodiumdode- saline solution (PSS) to a 2.5 ml volume, cylsulfate polyacrylamide gel electrophoretic were prepared. The anti-birtoxin antibody (SDS-PAGE) analysis according to Laemmli was prepared at doses ranging from 0.5 ml to (1970). Venom of A. crassicauda and P. 1.5 ml. On the other hand A. crassicauda transvaalicus scorpions were separated on venom (1 MLD) also prepared for each precast NuPAGE 12 % Bis-Tris Gel are mouse and mixed with 1.5 ml anti-birtoxin electrophoretically transferred to the antibody. The final volume all dilutions were nitrosellulose membrane (NCM) and divided made up to 5 ml with PSS. The solutions to two sections. The membranes were incu- were incubated for 60 min at room temper- bated in blocking buffer (3% BSA in TBST ature. Then, 0.5 ml of each solution was [0.1 Tween 20, 150 mM NaCl, 10 mM Tris- subcutaneously injected into groups of eight Cl, pH 7.5]) for one hour. The membranes mice previously injected with A. crassicauda were then washed three times with TBST venom. The control groups were only in- (Tris-Buffered Saline Tween-20, [0.1% jected with 1 MLD of the venom diluted in Tween 20, 150 mM NaCl, 10 mM Tris-Cl, PSS. The numbers of surviving mice were pH 7.4]) and strips of the membrane were recorded up to 48 h. After administration, exposed to pre-immune serum for each animals were monitored for 48 hours and the antivenom for 30 min followed by three number of living animals was recorded. The washes and incubated with antivenom of A. anti-birtoxin doses that prevented 100 % crassicauda (1: 4000) and the anti-birtoxin deaths in the groups were considered the Ab (1: 1000). Membranes were again washed minimum effective doses (MED). 3 times with TBST, and then incubated with A solution of A. crassicauda venom (3 horseradish peroxidase-conjugated anti-horse MLD for each mouse) diluted in physiologic antibody and HRP- conjugated anti-rabbit saline solution (PSS) to a final volume of 2.5 (1: 5000) for 60 min. The membranes were ml. The anti-birtoxin antibody was prepared washed with TBST for 10 minutes and anti- at doses ranging from 0.5 ml to 1.5 ml. Sep- gens were visualized using the Immun-Star arately, A. crassicauda venom (1 MLD, 62 HPR Chemiluminescent subtrate (BioRad). µl venom for each mouse) was prepared for Membranes were exposed to X-ray film in a each mouse and mixed with 1.5 ml anti- dark room and developed. birtoxin antibody. All solutions were then diluted to a final volume of 5 ml using PSS. Results These solutions were incubated for 60 min at room temperature. Then, 0.5 ml of each so- The MLD of the A. crassicauda venom lution was subcutaneously injected into groups was found to be 35 µg/20 g mouse (1.75 of eight mice previously injected with A. mg/kg) by sc injection route (Table 1). The crassicauda venom. The control groups were potency of A. crassicauda antivenom (500 only injected with 1 MLD of the venom di- μl) has previously been determined to be luted in PSS using the same volumes. Fol- neutralizing 2 MLD in 150g rats according lowing administrations the animals were mon- to manufacturer’s instructions. itored up to 48h and survival was noted. The Here this was confirmed to be the case. anti-birtoxin doses that prevented 100 % To assess the potency of the anti-birtoxin deaths in the groups were considered the min- antibody, increasing doses of the antibody imum effective doses (MED). were used while the amount of A. crassicauda 178 http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 176–183 SA V Zoelen et al.: Antigenic Cross-Reactivity …

venom was kept constant (1 and 3 MLD). The Ac antivenom (0.8 mL) potently neu- tralized 3 MLD of the venom while all control mice died. However, 1.5 ml of anti- birtoxin antibody was not able to neutralize even 1 MLD of venom of A. crassicauda scorpion (Table 2). Despite the lack of the ability of anti-birtoxin antibodies to neu- tralize the venom of Ac western blots in- dicate that there is a certain level of cross reactivity between this Ab and the Ac venom (Fig. 1). Although the peptide fall below 10 kDa molecular weight range often times on western blots a smear in the range of 5–15 kDa corresponds to these peptides and their heteromers due to the running conditions. As shown by Western blotting, A. crassicauda antivenom strongly reacted with the components of both P. transvaalicus and Fig. 1. The venoms from Androctonus crassicauda A. crassicauda venoms (Fig. 1). Similarly, (A) and Parabuthus transvaalicus (B) were separated the anti-birtoxin Ab strongly reacted with and transferred to membranes. Proteins were detected both P. transvaalicus and A. crassicauda using the RSHC anti-Ac (1: 4,000) on the panel I and scorpion venoms as well. Fig. 1 shows that anti-birtoxin Ab (1: 1,000) on the panel II. Molecular weight (M) markers on the panel II are SeeBlue® proteins that were detected using the anti- Plus2 (Invitrogen Corporation, USA) birtoxin Ab all fall under 19 kDa molecular mass.

Table 1. Minimum Lethal Dose of Androctonus crassicauda venom

Androctonus crassicauda scorpion Negative control Venom (μg/mouse) Mice (Death/total) PSS (μl/mouse) Mice (Death/total) 10 0/5 15 1/5 20 2/5 500 0/5 25 4/5 35* 5/5* MLD: 35 μg/ 20g mouse

Table 2. Neutralization capacity of the anti-birtoxin and antivenom was assayed for Androctonus crassicauda venom in mice

MED of Androctonus crassicauda antivenom MED of anti-birtoxin antibody Venom (μg/mouse) Antivenom (μl) Mice (Surviving /total) Venom (μg/mouse) Antibody (μl) Mice (Surviving /total) 105 400 5/8 105 500 0/8 105 800 8/8 105 1000 0/8 105 1000 8/8 105 1500 0/8 35 (Control) - 0/8 35 1500 0/8*

MED: 800 μl MED: No determined 179 http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 176–183 SA V Zoelen et al.: Antigenic Cross-Reactivity …

Discussion Venom effect on the autonomic nervous described as a neutralization unit ac- system cording their standards (Theakston et al. The antivenom therapy plays an important 2003, Ozkan et al, 2007). Therefore in the role in the treatment of scorpionism cases. present study, MLD and MED were deter- As several studies stated that upon poisoning mined instead of LD50 and ED50. In our caused by scorpion stings, it is often recom- study, the MED of the antivenom against 3 mended that the patients treated with spe- MLD A. crassicauda venom was 0.8ml while cies-specific antivenom and this must be ad- 1.5ml of anti-birtoxin antibody was not able ministered early upon envenomation (Alex- to neutralize even 1 MLD of the venom of A. ander 1984, El-Amin 1992, Ismail 1993, crassicauda scorpion. Therefore the MED of Sofer et al. 1994). the anti-birtoxin was not determined. The potentially dangerous and medically Inceoglu et al. (2001) determined that important scorpion species venom effect on native birtoxin from P. transvaalicus also the autonomic nervous system (Bawaskar has the average molecular mass of 6543.6 2005). Androctonus crassicauda is consid- Da. Besides, Martin-Eauclaire et al. (2005) ered as the most significant species of scor- described new members of birtoxin-like pions in Turkey and neighbouring countries peptides familiy from the venom of A. Iran, Iraq and Syria causing a large number austrailis. Moreover they notified that this of envenomations every year (Radmanesh new family might probably exist in other 1990, Ozkan et al. 2006b, Chippaux and “Old-World” Buthidae venoms (Martin- Goyffon 2008, Dehghani and Khamechian Eauclaire et al. 2005). Recently, this species 2008, Antopolsky et al. 2009, Bosnak et al. from Turkey has been studied and five 2009, Dehghani et al. 2009, Shahbazzadeh et toxins described by Calıskan et al. (2006) al. 2009, Dehghani and Fathi 2012). two of which (Acra 1, 6496.8 Da and Acra 2 Parabuthus species are medically the most 7848.6 Da), were lethal to mice. important scorpions in South Africa thus In this study, Western blotting showed Bergman (1997) reports on the clinical man- the presence of components from A. ifestations of human envenomation by P. crassicauda and P. transvaalicus scorpion transvaalicus and the incidence rate of venoms strongly cross react with the A. envenomation in Zimbabwe. crassicauda antivenom. However, Western Krifi et al. (1998) reported the difficulties blotting of the A. crassicauda scorpion in standardization the venom quality and venom using the RHSC generated antibody LD50 determination which are partly related showed that not all the venom components to geographical origin, the age of the ven- cross reacted with the anti-birtoxin antibody. omous species, the season and venom ex- The antibodies only cross reacted with traction procedures, the number of speci- components falling under the 19 kDa protein mens milked, the breeding conditions, and size of A. crassicauda venom. This is not also the species’ strain, of the test animal unexpected since most of the neurotoxic body weight and administration route. These peptide components fall under this range. parameters must be accepted as important However these findings indicate that in factors for standardization of the venom contrast to the P. transvaalicus venom, in A. toxicity and the antivenom efficacy. Thus, crassicauda venom the birtoxin like peptides potency of antivenom is estimated by na- contribute minimally to the . tional or regional control authority and is This reiterates the fact that species differ-

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ences and geographical variations which Antopolsky M, Salameh S, Stalnikowicz R result in a diverse number of neurotoxic (2009) Need for emergency depart- peptides in the venom needs to be consid- ment observation after scorpion sting: ered as an important factor in developing prospective study and review of the antivenoms that are effective. The finding literature in the Middle East. Eur J that P. transvaalicus venom can be neutral- Emerg Med. 16: 206–208. ized with a polyclonal antibody raised against Balozet L (1971) Scorpionism in the Old the first 18 amino acid residues of birtoxin World. In: Bücherl W, Buckley E (Eds) seems to be an exception (Inceoglu et al. Venomous Animals and their Venoms. 2006) though it remains to be seen if horse Volume 3. Venomous Intervertebrates. derived antivenom against A. crassicauda will Academic Press, New York, pp. 349– neutralize the venom of P. transvaalicus. Simi- 371. larly, it remains to be seen if the anti-birtoxin Bawaskar HS (2005) Management of severe antibodies will neutralize the venom of closely scorpion sting at rural settings: what is related species including P. granulatus. the role of scorpion antivenom? J Venom Anim Toxins Incl Trop Dis. 11: 3–7. Conclusion Bergman NJ (1997) Clinical description of Parabuthus transvaalicus scorpionism in Zimbabwe. Toxicon. 35: 759 771. The bioassays and Western blotting of A. – Bosnak M, Ece A, Yolbas I, Bosnak V, crassicauda venom with the anti-birtoxin Kaplan M, Gurkan F (2009) Scorpion antibodies produced against a synthetic pep- sting envenomation in children in South- tide showed that these antibodies cross re- east Turkey. Wilderness Environ Med. acted but did not neutralize the venom of A. 20: 118 124. crassicauda. – Bücherl W (1971) Classification, Biology and Venom Extraction of Scorpion. In: Acknowledgements Bücherl W, Buckley E (Eds) Venom- ous Animals and their Venoms. Vol- This project was supported by Refik ume 3. Venomous Intervertebrates. Ac- Saydam Hygiene Center of Ministry of ademic Press, New York, pp. 317–347. Health, Ankara, Turkey. We are grateful to Caliskan F, García BI, Coronas FI, Batista the Directory of Refik Saydam Public Health CV, Zamudio FZ, Possani LD (2006) Agency. We also thank to Dr Bruce D Ham- Characterization of venom compo- mock and all Hammock Laboratory (Depart- nents from the scorpion Androctonus ment of Entomology, University of Califor- crassicauda of Turkey: peptides and nia, USA) members for their support and genes. Toxicon. 48: 12–22. help. The authors declare that there is no Chippaux JP, Goyffon M (2008) Epidemi- conflict of interests. ology of scorpionism: A global ap- praisal. Acta Trop. 107(2): 71–79. References Dehghani R, Fathi B (2012) Scorpion sting in Iran: A review. Toxicon. 60: 919– Alexander JOD (1984) Scorpion Stings. In: 933. Alexander JOD (Ed) Arthropods and Dehghani R, Djadid ND, Shahbazzadeh D, Human Skin. Springer-Verlag, Berlin, Bigdelli S (2009) Introducing p. 199. Compsobuthus matthiesseni (Birula, 1905) scorpion as one of the major 181 http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 176–183 SA V Zoelen et al.: Antigenic Cross-Reactivity …

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