most of the medically important scorpion species belong to buthus , parabuthus , mesobuthus , tityus , leiurus , androctonus and centruroides genera of the buthidae family ( balozet 1971 , bcherl 1971 , efrati 1978 ) .
scorpion venoms can be classified into two groups according to their molecular sizes , long - chain and short - chain neurotoxins .
the short - chain neurotoxins are 3,000 to 4,400 da and act on potassium or chloride channels .
long - chain neurotoxins are 6,500 to 7,800 da and act mostly on sodium channels ( possani et al . 1999 , 2000 ,
it has been estimated that 100.000 distinct peptides exist in scorpion venom but only limited number of these peptides have been described ( possani et al . 1999 , 2000 , martin - eauclaire et al .
the unique specific treatment of scorpion envenomations is immunotherapy with antibodies from immunized horses ( ghalim et al .
2000 ) . however , the venom is a complex mixture of antigens wherein not all components are equally important for the production of neutralizing antibodies .
thus , the identification of immunogenic protein(s ) and/or their neutralizing epitopes may lead to the use of more clearly defined substances as immunogens to develop efficient antivenoms or to their use as antigens .
the venom of p. transvaalicus consists of recently described closely related neurotoxins named birtoxin family ( inceoglu et al . 2001 , 2005 ) .
an antibody developed using a synthethic peptide composed of the first 18 amino acid residues of birtoxin displayed strong reactivity with the whole venom of p. transvaalicus , p. leisoma and pure birtoxin ( inceoglu et al .
these antibodies also neutralized the venom of p. transvaalicus in mice . recently , calkan et al .
( 2006 ) also reported the presence of peptides in a. crassicauda venom that belong to the birtoxin - like peptide family . in this study , we tested the anti - birtoxin antibodies for their ability to neutralize the lethal effects of a. crassicauda scorpion venom .
venom was obtained from mature a. crassicauda scorpions ( from sanliurfa ) by electrical stimulation of the telson .
the venom was mixed with sterile double - distilled water and centrifuged at 15,000 rpm for 15 min at 4 c .
the supernatant was immediately lyophilized at refik saydam public health agency ( rspha ) and stored at 80c until use .
venom of commercially obtained p. transvaalicus scorpions were collected as described ( inceoglu et al .
briefly , increasing venom doses , mixed half - and - half with adjuvants , were injected subcutaneously into horses on the 1 , 14 , 21 , 28 
 , 35 and 42 days . on the 45 , 48 and 51 , 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 .
one dose of rsha anti - ac was normalized to neutralize 2 mld of a. crassicauda venom in rats when tested subcutaneously .
the 18 residues n - terminal portion of birtoxin - like peptides  nh2-advpgnypld kdgntykc was commercially synthesized by sigma and polyclonal antibodies against this peptide were raised by sigma - genosys ( inceoglu et al .
briefly , the synthetic peptide was cross - linked to keyhole limpet hemocyanin and rabbits were immunized .
the bleedings were done after the 4 , 5 and 6 booster doses and pooled .
igg molecules were purified using a protein a antibody purification kit from sigma following the manufacturer s instructions .
protein concentrations were determined using a bca kit ( pierce , usa ) with ovalbumin as the standard .
all the experiments were performed according to the guidelines by the ethical committee of the faculty of veterinary medicine in ankara university .
the minimum lethal dose ( mld ) of venom was assessed by subcutaneously ( sc ) injections in mice ( 202 g ) .
five mice per each dose - group were injected sc with doses of venom , diluted in 0.5 ml saline solution .
an equivalent volume of 0.5 ml saline was injected into five mice as negative control group .
the animals were observed for 48 h after venom injection in order to determine mld .
a solution of a. crassicauda venom ( 3 mld for each mouse ) diluted in physiologic saline solution ( pss ) to a 2.5 ml volume , were prepared .
the anti - birtoxin antibody was prepared at doses ranging from 0.5 ml to 1.5 ml . on the other hand a. crassicauda venom ( 1 mld ) also prepared for each mouse and mixed with 1.5 ml anti - birtoxin antibody .
, 0.5 ml of each solution was subcutaneously injected into groups of eight mice previously injected with a. crassicauda venom .
the control groups were only injected with 1 mld of the venom diluted in pss .
the numbers of surviving mice were recorded up to 48 h. after administration , animals were monitored for 48 hours and the number of living animals was recorded .
the anti - birtoxin doses that prevented 100 % deaths in the groups were considered the minimum effective doses ( med ) .
a solution of a. crassicauda venom ( 3 mld for each mouse ) diluted in physiologic saline solution ( pss ) to a final volume of 2.5 ml .
the anti - birtoxin antibody was prepared at doses ranging from 0.5 ml to 1.5 ml .
separately , a. crassicauda venom ( 1 mld , 62 l venom for each mouse ) was prepared for each mouse and mixed with 1.5 ml anti - birtoxin antibody .
these solutions were incubated for 60 min at room temperature . then , 0.5 ml of each solution was subcutaneously injected into groups of eight mice previously injected with a. crassicauda venom .
the control groups were only injected with 1 mld of the venom diluted in pss using the same volumes .
the anti - birtoxin doses that prevented 100 % deaths in the groups were considered the minimum effective doses ( med ) .
venoms were analyzed by sodiumdodecylsulfate polyacrylamide gel electrophoretic ( sds - page ) analysis according to laemmli ( 1970 ) .
venom of a. crassicauda and p. transvaalicus scorpions were separated on precast nupage 12 % bis - tris gel are electrophoretically transferred to the nitrosellulose membrane ( ncm ) and divided to two sections .
the membranes were incubated in blocking buffer ( 3% bsa in tbst [ 0.1 tween 20 , 150 mm nacl , 10 mm tris - cl , ph 7.5 ] ) for one hour .
the membranes were then washed three times with tbst ( tris - buffered saline tween-20 , [ 0.1% tween 20 , 150 mm nacl , 10 mm tris - cl , ph 7.4 ] ) and strips of the membrane were exposed to pre - immune serum for each antivenom for 30 min followed by three washes and incubated with antivenom of a. crassicauda ( 1 : 4000 ) and the anti - birtoxin ab ( 1 : 1000 ) .
membranes were again washed 3 times with tbst , and then incubated with horseradish peroxidase - conjugated anti - horse antibody and hrp - conjugated anti - rabbit ( 1 : 5000 ) for 60 min .
the membranes were washed with tbst for 10 minutes and antigens were visualized using the immun - star hpr chemiluminescent subtrate ( biorad ) .
venom was obtained from mature a. crassicauda scorpions ( from sanliurfa ) by electrical stimulation of the telson .
the venom was mixed with sterile double - distilled water and centrifuged at 15,000 rpm for 15 min at 4 c .
the supernatant was immediately lyophilized at refik saydam public health agency ( rspha ) and stored at 80c until use .
venom of commercially obtained p. transvaalicus scorpions were collected as described ( inceoglu et al .
briefly , increasing venom doses , mixed half - and - half with adjuvants , were injected subcutaneously into horses on the 1 , 14 , 21 , 28 
 , 35 and 42 days . on the 45 , 48 and 51 , 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 .
one dose of rsha anti - ac was normalized to neutralize 2 mld of a. crassicauda venom in rats when tested subcutaneously .
the 18 residues n - terminal portion of birtoxin - like peptides  nh2-advpgnypld kdgntykc was commercially synthesized by sigma and polyclonal antibodies against this peptide were raised by sigma - genosys ( inceoglu et al .
briefly , the synthetic peptide was cross - linked to keyhole limpet hemocyanin and rabbits were immunized .
the bleedings were done after the 4 , 5 and 6 booster doses and pooled .
igg molecules were purified using a protein a antibody purification kit from sigma following the manufacturer s instructions .
protein concentrations were determined using a bca kit ( pierce , usa ) with ovalbumin as the standard .
all the experiments were performed according to the guidelines by the ethical committee of the faculty of veterinary medicine in ankara university .
the minimum lethal dose ( mld ) of venom was assessed by subcutaneously ( sc ) injections in mice ( 202 g ) .
five mice per each dose - group were injected sc with doses of venom , diluted in 0.5 ml saline solution .
an equivalent volume of 0.5 ml saline was injected into five mice as negative control group .
the animals were observed for 48 h after venom injection in order to determine mld .
a solution of a. crassicauda venom ( 3 mld for each mouse ) diluted in physiologic saline solution ( pss ) to a 2.5 ml volume , were prepared .
the anti - birtoxin antibody was prepared at doses ranging from 0.5 ml to 1.5 ml . on the other hand a. crassicauda venom ( 1 mld ) also prepared for each mouse and mixed with 1.5 ml anti - birtoxin antibody . the final volume all dilutions
the solutions were incubated for 60 min at room temperature . then , 0.5 ml of each solution was subcutaneously injected into groups of eight mice previously injected with a. crassicauda venom .
the control groups were only injected with 1 mld of the venom diluted in pss .
the numbers of surviving mice were recorded up to 48 h. after administration , animals were monitored for 48 hours and the number of living animals was recorded .
the anti - birtoxin doses that prevented 100 % deaths in the groups were considered the minimum effective doses ( med ) .
a solution of a. crassicauda venom ( 3 mld for each mouse ) diluted in physiologic saline solution ( pss ) to a final volume of 2.5 ml .
the anti - birtoxin antibody was prepared at doses ranging from 0.5 ml to 1.5 ml .
separately , a. crassicauda venom ( 1 mld , 62 l venom for each mouse ) was prepared for each mouse and mixed with 1.5 ml anti - birtoxin antibody .
these solutions were incubated for 60 min at room temperature . then , 0.5 ml of each solution was subcutaneously injected into groups of eight mice previously injected with a. crassicauda venom .
the control groups were only injected with 1 mld of the venom diluted in pss using the same volumes . following administrations
the anti - birtoxin doses that prevented 100 % deaths in the groups were considered the minimum effective doses ( med ) .
venoms were analyzed by sodiumdodecylsulfate polyacrylamide gel electrophoretic ( sds - page ) analysis according to laemmli ( 1970 ) .
venom of a. crassicauda and p. transvaalicus scorpions were separated on precast nupage 12 % bis - tris gel are electrophoretically transferred to the nitrosellulose membrane ( ncm ) and divided to two sections .
the membranes were incubated in blocking buffer ( 3% bsa in tbst [ 0.1 tween 20 , 150 mm nacl , 10 mm tris - cl , ph 7.5 ] ) for one hour .
the membranes were then washed three times with tbst ( tris - buffered saline tween-20 , [ 0.1% tween 20 , 150 mm nacl , 10 mm tris - cl , ph 7.4 ] ) and strips of the membrane were exposed to pre - immune serum for each antivenom for 30 min followed by three washes and incubated with antivenom of a. crassicauda ( 1 : 4000 ) and the anti - birtoxin ab ( 1 : 1000 ) .
membranes were again washed 3 times with tbst , and then incubated with horseradish peroxidase - conjugated anti - horse antibody and hrp - conjugated anti - rabbit ( 1 : 5000 ) for 60 min .
the membranes were washed with tbst for 10 minutes and antigens were visualized using the immun - star hpr chemiluminescent subtrate ( biorad ) .
the mld of the a. crassicauda venom was found to be 35 g/20 g mouse ( 1.75 mg / kg ) by sc injection route ( table 1 ) .
the potency of a. crassicauda antivenom ( 500 l ) has previously been determined to be neutralizing 2 mld in 150 g rats according to manufacturer s instructions
. minimum lethal dose of androctonus crassicauda venom here this was confirmed to be the case . to assess the potency of the anti - birtoxin antibody , increasing doses of the antibody were used
while the amount of a. crassicauda venom was kept constant ( 1 and 3 mld ) . the ac antivenom ( 0.8 ml ) potently neutralized 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 neutralize the venom of ac western blots indicate that there is a certain level of cross reactivity between this ab and the ac venom ( fig .
although the peptide toxins fall below 10 kda molecular weight range often times on western blots a smear in the range of 515 kda corresponds to these peptides and their heteromers due to the running conditions .
the venoms from androctonus crassicauda ( a ) and parabuthus transvaalicus ( b ) were separated and transferred to membranes .
proteins were detected using the rshc anti - ac ( 1 : 4,000 ) on the panel i and anti - birtoxin ab ( 1 : 1,000 ) on the panel ii . molecular weight ( m )
markers on the panel ii are seeblue plus2 ( invitrogen corporation , usa ) neutralization capacity of the anti - birtoxin and antivenom was assayed for androctonus crassicauda venom in mice as shown by western blotting , a. crassicauda antivenom strongly reacted with the components of both p. transvaalicus and a. crassicauda venoms ( fig .
similarly , the anti - birtoxin ab strongly reacted with both p. transvaalicus and a. crassicauda scorpion venoms as well .
1 shows that proteins that were detected using the anti - birtoxin ab all fall under 19 kda molecular mass .
as several studies stated that upon poisoning caused by scorpion stings , it is often recommended that the patients treated with species - specific antivenom and this must be administered early upon envenomation ( alexander 1984 , el - amin 1992 , ismail 1993 , sofer et al .
the potentially dangerous and medically important scorpion species venom effect on the autonomic nervous system ( bawaskar 2005 ) .
androctonus crassicauda is considered as the most significant species of scorpions in turkey and neighbouring countries iran , iraq and syria causing a large number of envenomations every year ( radmanesh 1990 , ozkan et al .
2006b , chippaux and goyffon 2008 , dehghani and khamechian 2008 , antopolsky et al .
parabuthus species are medically the most important scorpions in south africa thus bergman ( 1997 ) reports on the clinical manifestations of human envenomation by p. transvaalicus and the incidence rate of envenomation in zimbabwe .
( 1998 ) reported the difficulties in standardization the venom quality and ld50 determination which are partly related to geographical origin , the age of the venomous species , the season and venom extraction procedures , the number of specimens milked , the breeding conditions , and also the species strain , of the test animal body weight and administration route .
these parameters must be accepted as important factors for standardization of the venom toxicity and the antivenom efficacy .
thus , potency of antivenom is estimated by national or regional control authority and is described as a toxin neutralization unit according their standards ( theakston et al . 2003 , ozkan et al , 2007 ) .
therefore in the present study , mld and med were determined instead of ld50 and ed50 . in our study , the med of the antivenom against 3 mld a. crassicauda venom was 0.8 ml while 1.5 ml of anti - birtoxin antibody was not able to neutralize even 1 mld of the venom of a. crassicauda scorpion .
( 2001 ) determined that native birtoxin from p. transvaalicus also has the average molecular mass of 6543.6 da . besides , martin - eauclaire et al .
( 2005 ) described new members of birtoxin - like peptides familiy from the venom of a. austrailis .
recently , this species from turkey has been studied and five toxins described by calskan et al .
( 2006 ) two of which ( acra 1 , 6496.8 da and acra 2 7848.6 da ) , were lethal to mice . in this study ,
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 rhsc 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 .
this is not unexpected since most of the neurotoxic peptide components fall under this range .
however these findings indicate that in contrast to the p. transvaalicus venom , in a. crassicauda venom the birtoxin like peptides contribute minimally to the neurotoxicity .
this reiterates the fact that species differences and geographical variations which result in a diverse number of neurotoxic peptides in the venom needs to be considered as an important factor in developing antivenoms that are effective .
the finding that p. transvaalicus venom can be neutralized with a polyclonal antibody raised against the first 18 amino acid residues of birtoxin seems to be an exception ( inceoglu et al .
2006 ) though it remains to be seen if horse derived antivenom against a. crassicauda will neutralize the venom of p. transvaalicus .
similarly , it remains to be seen if the anti - birtoxin antibodies will neutralize the venom of closely related species including p. granulatus .
as several studies stated that upon poisoning caused by scorpion stings , it is often recommended that the patients treated with species - specific antivenom and this must be administered early upon envenomation ( alexander 1984 , el - amin 1992 , ismail 1993 , sofer et al .
the potentially dangerous and medically important scorpion species venom effect on the autonomic nervous system ( bawaskar 2005 ) .
androctonus crassicauda is considered as the most significant species of scorpions in turkey and neighbouring countries iran , iraq and syria causing a large number of envenomations every year ( radmanesh 1990 , ozkan et al .
2006b , chippaux and goyffon 2008 , dehghani and khamechian 2008 , antopolsky et al .
parabuthus species are medically the most important scorpions in south africa thus bergman ( 1997 ) reports on the clinical manifestations of human envenomation by p. transvaalicus and the incidence rate of envenomation in zimbabwe .
( 1998 ) reported the difficulties in standardization the venom quality and ld50 determination which are partly related to geographical origin , the age of the venomous species , the season and venom extraction procedures , the number of specimens milked , the breeding conditions , and also the species strain , of the test animal body weight and administration route .
these parameters must be accepted as important factors for standardization of the venom toxicity and the antivenom efficacy .
thus , potency of antivenom is estimated by national or regional control authority and is described as a toxin neutralization unit according their standards ( theakston et al . 2003 , ozkan et al , 2007 ) .
therefore in the present study , mld and med were determined instead of ld50 and ed50 . in our study , the med of the antivenom against 3 mld a. crassicauda venom was 0.8 ml while 1.5 ml of anti - birtoxin antibody was not able to neutralize even 1 mld of the venom of a. crassicauda scorpion .
( 2001 ) determined that native birtoxin from p. transvaalicus also has the average molecular mass of 6543.6 da . besides , martin - eauclaire et al .
( 2005 ) described new members of birtoxin - like peptides familiy from the venom of a. austrailis .
recently , this species from turkey has been studied and five toxins described by calskan et al .
( 2006 ) two of which ( acra 1 , 6496.8 da and acra 2 7848.6 da ) , were lethal to mice . in this study ,
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 rhsc 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 .
this is not unexpected since most of the neurotoxic peptide components fall under this range .
however these findings indicate that in contrast to the p. transvaalicus venom , in a. crassicauda venom the birtoxin like peptides contribute minimally to the neurotoxicity .
this reiterates the fact that species differences and geographical variations which result in a diverse number of neurotoxic peptides in the venom needs to be considered as an important factor in developing antivenoms that are effective .
the finding that p. transvaalicus venom can be neutralized with a polyclonal antibody raised against the first 18 amino acid residues of birtoxin seems to be an exception ( inceoglu et al .
2006 ) though it remains to be seen if horse derived antivenom against a. crassicauda will neutralize the venom of p. transvaalicus .
similarly , it remains to be seen if the anti - birtoxin antibodies will neutralize the venom of closely related species including p. granulatus .
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 .
 background : antivenom is still widely used in the treatment of envenomation as there are no vaccines or other effective agents available against animal venoms . 
 recently , neurotoxins named birtoxin family have been described from parabuthus transvaalicus and androctonus crassicauda . 
 the aim of the present study was to test the anti - birtoxin antibodies for their ability to neutralize the lethal effects of a. crassicauda scorpion 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 g/ 20 g 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 . 
Introduction
Materials and Methods
Venoms
Antivenom (RSHC anti-Ac)
Anti-birtoxin antibody
Determination of the Minimum Lethal Dose (MLD) in mice
Serum-neutralization assays in mice
Gel electrophoresis of the venoms and Western Blotting
Results
Discussion
Venom effect on the autonomic nervous system
Conclusion