- Email: [email protected]
Immunological studies with scorpion (Androctonus amoreuxi Aud. & Sav.) venom
Tostcaa, 1975, Vol . 13, PP" 405--108 . Per~mon Pies. Printed In l3reat Hrltain.
IMMUNOLOGICAL STUDIES WITH SCORPION
(ANDROCTONUS AMOREUXI AUD. & SAV.) VENOM M.
ISMAIL, A. GHnzAL, M. F. EL-ASMAR * and A. A. AsnEh-RAxMAx Department of Pharmacology, Faculty of Pharmacy, University of Alexandria, Egypt (Accepted jor publication 14 May 1975) M. IsiHwlt., A. GI;"~"~, M. F. EL-AsMax and A. A. AHn~RexMerl. immunological studies with scorpion (Androctonus amoretexl Aud. & Sav.) venom. Toxlcon 13, 405-408, 1975 . An antiveninwasprepared forA. amoreuxi venom by hyper-immunizing rabbits. The antivenin protested mice against the lethal action of the venom and prevented blockade of twitch activity caused by the venom in both the phrenic nerve-hetnidiaphragm of the rat and the cat tibialis anterior preparation . The antivenin also prevented the cardiac stimulant and the uterine inhibitory effects of the venom . Using immunodiffusion and immuncelectrophoresis techniques, it was possible to reveal 7 prominentand 2faintprecipitin bandswith A. amoreuxi venom. Precipitin bands corresponding to individual venom fractions were also identified. Antigenic components were also found with venoms from the scorpions Bathos minax, Buthus occitaJUU and Lelurus quinquestriatus . The bands found, however, were not identical with those of A. amoreuxi venom. INTRODUCTION
Androctonus amoreuxi is widely distributed in Egypt and neighbouring countries (BALOZSr, 1971). No specific antivenin is available in Egypt against its venom despite the frequency of the scorpion stings . Individuals subjected to A. amoreuxi stings are usually treated with a poly antivenin prepared from scorpion venoms but not including A. atnoreuxi. BALOZET (1971), however, pointed out that "scorpion antivenins are rather specific". In a previous publication from this laboratory (GxAZa~l, et al., 1975), the venom from A. amoreuxi was found to possess potent neurotoxic and cardiotoxic properties. It was thought worthwhile, therefore, to attempt the preparation of a specific antivenin for A. amoreuxi and to test its protecting properties in experimental animals. TrIE SCORPION
MATERIALS AND METHODS
Venom was obtained from mature A. amoreuxl as described by Ist~,n, et al. (1973). Hyperimmunization of rabbits Male rabbits weighing between 2 and 2" 5 kg were kept in the animal house for 2 weeks before immunization . The rabbits were tested for natural immunity and only those whose serum showed no precipitin bands with A. amoreuxt venom were used. The rabbits were injected subcutaneously with A. amorercxivenom, each dose being emulsified in 0" 5 ml complete Freund's adjuvant (Difco Laboratories, Detroit, Mich .), according to the following schedule : two injections of 200 pg each in the first week, three injections of 300 Itg each in the second week and one igjection of 500 Etg in each of the fifth and the subsequent S weeks. Five days after the final dose, blood was withdrawn from tho marginal ear veins and the serum tested for positive precipitin bands. Rabbits showing positive precipitin bands were injected with 500 !ag venom and were bled 6 days later. The collected blood was left at 25 °C for 2 hr to form a spontaneous clot. Serum was obtained by centrifugation at 6000 rev per min, pooled, and thiomerosal (1 : 10,000) was added as a preservative. The serum was then distributed in small tubes and kept at -20°C until used. *Department of Biochemistry, Faculty of Medicine, Ain Shams University, Cairo, Egypt. TOXJCON 1975 V°t. J3
404
406
M. ISMAIL, A. GHAZAL, M . F. EL-ASMAR and A. A. ABDEL-RAHMAN
Immuirochemkal studies Electrophoresis of the crude venom was carried out as described by Gxnznr. et al. (197 . Fractions separated by cellulose acetate electrophoresis were eluted with 0'3 ~ NaCI solution and adjusted to a final concentration of 1 mg per ml . Immunodiffusion experiments were carried out on lantern slides (5 x 5 cm) using 1'2~ Noble agar (Difco Lab., Detroit, Mich.) in 0~9~ NaCI solution. Sodium azide in a concentration of 005 ~ was added to retard bacterial growth. Double diffusion experiments were carried out as described by OUCFITERLONY (1948). The wells were filled with 20 ul volumes of either the crude venom, the various fractions separated by cellulose acetate electrophoresis or the antiserum. After developing of the precipitin bands (48 hr at 25°C), the plates were washed for 24 hr in saline, dried and stained with amidoschwartt 10 B (0~5 ~ in 5 acetic acid) for 7 min, washed with methanol : acetic acid (9 : 1), dried in air and photographed . Immunoelectrophoresis was carried out by the transfer method (KoxN, 1968) using lantern slides (5 x 20 cm) covered with agar. A pattern indicating the desired relative positions of the electrophoretic and antivenom strips was drawn on white paper. This pattern was clearly visible through the slide when it was placed underneath . The electrophoresic run of the venom (50 ul samples of a 20 mg per ml solution) was carried out on cellulose acetate strips (50 x 200 mm) using barbitone buffer, 005 ionic strength, pH 8~6 for 150 min and applying 25 V per cm . At the end of the run, the strips were cut longitudinally into three equal parts. The middle third was dried in hot air and stained with Ponceau S (0'2 % in 3 ~ trichloroacetic acid) and used to locate the protein bands on the other two unstained thirds. Afilter paper strip (Whatman No . 3, approximately 2 mm wide) was impregnated with the antivenin (80 pl) and placed on the gel at the previously indicated position . One third of thecellulose acetate strip was placed at one side of the antivenom strip. The bands on the remaining third were cut out and placed, each 0~5 cm apart, at the other side of the antivenin strip. The plates were then placed in a moist chamber at room temperature (25°C) and diffusion was carried out for 248 hr. The slides were then washed in 0~9~ NaCI solution, dried and stained with Ponceau S, washed with 5 ~ acetic acid, dried in air and then photographed . Neutralization tests In these experiments A. amoreuxi venom was incubated with the specific antivenin for 1 hr at 37°C in the proportion of 1 mg venom to 4 ml serum. (a) Protection against venom lethality in mice . The venom-antivenin mixture was shaken and a dose equal to the tn bo (088 mg per kB, G><;az~L et al., 1975) was injected intramuscularly into each of 10 mice. Doses of the incubated mixture equal to 2-5 times the ~su were injected each into a group of 10 mice . All mice were observed for 24 hr after injection. (b) Protection against thepharmacological effects of the venonr. In these experiments doses of the venomantivenin incubated mixture equivalent to doses ofthevenomshown to producepronounced pharmacological effects were tested on the following preparations : the isolated phrenic nerve"-hemidiaphragm of the rat (BÜLHRING, 1946), the cat tibialis anterior muscle (Bxowx, 1938), the isolated rabbit and guinea pig hearts (LangendorlF preparation) and the spontaneously contracting rat uterus in Ringer-Locke solution. RESULTS
In the immunodiffusion experiments precipitin bands were obtained with venom concentrations as low as 50 ltg per ml. However, in most of the experiments in both the immunodiffusion and the immunoelectrophoresis, a venom concentration of 20 mg per ml was used. Six prominent and one faint precipitin bands were shown with crude A. amoreuxi venom (Fig. 1) using the immunodiffusion technique. In some experiments, some of the bands were fused together. Precipitin bands were also revealed with the first four venom fractions closest to the origin . Thus each of fractions 1 and 2 revealed one precipitin band and each of fractions 3 and 4 showed three precipitin bands (Fig. 1). Testing the eluates of the areas between the different venom fractions showed a precipitin band to be revealed only by the area between fractions 3 and 4. This band cross-reacts with one of the precipitin bands formed by fraction 3. In the transfer immuncelectrophoresis technique five prominent precipitin bands were shown, while in the cut-transfer immunoelectrophoresis 7 prominent and 2 faint precipitin bands were revealed (Fig. 2). In contrast to the immunodiffusion technique precipitin bands, corresponding to the fraction at the origin, were found in the transfer immunoTOXICON 1975 Vol. 13
FIG . I . DOUBLE DIFFUSION REACTION OF RABBIT SERUM ANTIVENIN FOR A . a1Y.ore1(Xi VENOM (CENTRAL WELL) . V, A . amoreuxi venom (20 mg per ml); Or, band at the origin (1 mg per ml) ; F,~ ( l mg per ml),
the first 5 fractions in sequence from the origin ; separated by cellulose acetate electrophoresis . FIG . 2. IMMUNOELECTROPHORETIC PATTERN OF RABBIT SERUM VENOM (IN TROUGH).
ANTIVENIN FCR A . ali:Ofel(Xl
Or, band at the origin (1 mg per ml); FIB (1 mg per ml), the first 5 fractions in sequence from the origin ; separated by cellulose acetate electrophoresis. Upper trace, 'cut-transfer' immuncelectrophoresis; lower trace, transfer immuncelectrophoresis . Experimental conditions as in text . FIG . 3 . DOUBLE DIFFUSION REACTION OF RABBIT SERUM ANTIVENIN FOR A . QIY.'orEGXi VENOM (CENTRAL WELL) WITH OTHER SCORPION VENONS . A, A . atroreuxi venom ; Bm, Buthus minax venom ; Bo, Buthus occitanus venom ; Lq, Leiurus
yuirryuestriatus venom. Venom concentration 20 mg per ml .
TOX/CON 1975 Vol. l3
f.p . 406
FIG. 4. EFFECT OF INCUBATING A. pIY.O/PGXl VENOM WITH RABBIT SERUM ANTIVENIN ON THE RESPONSE OF THE RAT PHRENIC NERVE-HEMIDIAPHRAGM PREPARATION.
Rat phrenic nerve-hemidiaphragms in Krebs-Henseleit solution stimulated at 0~1 Hz . Bath capacity 30 ml . (a) : ", A. amoreuxt venom (300 ug). (b) : ",rabbit serum antivenin (12 ml); ", A . amoreuri venom (300 llg) -i rabbit serum antivenin (1 ~2 ml).
TOXlCON J975 Vol. !?
FIG, S . EFFECT OF INCUBATING A. ü,')IOICüSl VENOM WITH RABBIT SERUM ANTIVENIN ON THE RESPONSE OF THE CAT TIBIALIS ANTERIOR MUSCLE PREPARATION.
Male cat (4 kg) under chloralose anaesthesia. Maximal twitches of the tibialis anterior muscle elicited by stimulation of the nerve once every 10 sec. ", Venom (250 ug)r antivenin (1 ml); O, venom (250 ug). Injections were given close-arterially.
FIG. G. EFFECT OF INCUBATING A, arifOIEüX! VENOM WITH RABBIT SERUM ANTIVENIN ON THE RESPONSE OF THE SPONTANEOUSLY CONTRACTING RAT UTERUS .
Uterus in natural oestrus. Bath capacity 30 ml . ", Venom (60 ug); ", rabbit serum antivenin (0'3 ml) ; O, venom (60 Ilg) + rabbit serum antivenin (0~3 ml) ; W, wash. Contractions magnified 4 times under a constant load of 2 g using an auxotonic lever. TOXICON 1975 Vol. J3
Scorpion Venom Antivenin
407
electrophoresis (Fig. 2). In addition a precipitin band corresponding to fraction 5 was shown in the cut-transfer immuncelectrophoresis technique (Fig. 2). Prominent precipitin bands were also shown with venoms from Bathos minax (2 bands), Bathos occitam~s (3 bands) and Leiurus quinquestriatus (3 bands) using the immunodiffusion technique and venom concentration of 20 mg per ml (Fig. 3). The bands, however, were not identical with those of A . amoreuxi venom (Fig. 3). Incubation of A, amoreuxi venom with the specific antivenin protected the mice against the lethal action of a dose of the venom equivalent to double the LDsu. It also protected 80 and 60 % of the mice against the lethal action of doses of the venom equivalent to 4 and 5 times the I,n b n dose, respectively . The antivenin markedly attenuated the increase in twitch height of the rat phrenic nerve-hemidiaphragm by the venom (Fig. 4) and prevented the venom-induced contracture of the cat tibialis anterior muscle (Fig. ~. In both preparations the subsequent blockade of twitch activity was abolished. The antivenin also abolished the uterine inhibitory effect of the venom on the spontaneously contracting rat uterus (Fig. 6), and the initial negative isotropic and chronotropic effects and the subsequent marked positive isotropic effect on the isolated rabbit and guinea pig hearts . DISCUSSION
Fractionation and pharmacological investigations of A. amoreuxl venom were carried out by Gxnzni, et al. (197 . The venom was shown to possess potent neurotoxic and cardiotoxic properties and was fractionated by cellulose acetate electrophoresis at pH 8~6 into 8 cathodic fractions. Lethality and pharmacological activities were associated with only 3 venom fractions. Of these fractions, fraction 4 was the only lethal fraction and in addition it caused cardiac stimulation, uterine relaxation and an increase in the twitch height of the rat phrenic nerve-hemidiaphragm. Fraction 3 caused uterine relaxation and increased twitch height of the rat phrenic nerve-hemidiaphragm. Fraction 5 was only active on the cat tibialis anterior muscle causing contracture followed by blockade of twitch activity. The other fractions were without effect . In the present study the antivenin prepared against A . amoreuxi venom by hyperimmunizing rabbits protected the mice against the lethal action ofthe venom. This effect could be explained by the ability of the antivenin to neutralize the effects of fraction 4, the only lethal fraction. The formation of precipitin bands against fractions 3-5 could explain the protecting activity of the antivenin against the uterine inhibitory, the cardiac stimulant and the contracture and blockade of twitch activity of muscle preparations produced by the venom. The formation of more than one precipitin band with fraction 4 indicates that this fraction is probably composed of more than one protein. Attempts are being made to further purify this fraction . The use of `cut-transfer' immunoelectrophoresis, in which the individual fractions are cut out and placed, each 0~5 cm apart, on the side of the antivenin strip, offered better differentiation and identification of the precipitin bands than with the ordinary immunoelectrophoresis or immunodiffusion techniques . In addition, precipitin bands corresponding to the individual fractions could be recognized and used in explainingthe protecting activities of the antivenin. GLEnx et al. (1962) have investigated the intergeneric relationships of venoms obtained from scorpions separated by geographical barriers and showed that the venoms contained certain antigenic components in common. Also, Parrm and Nox~t (1962) showed that a large number of antigenic components are shared by members of the families Buthidae and Vejovidae . Using the immunodiffusion technique and A . amoreuxi antivenin, antigenic TOXICON 1975 Vol. l3
408
M. ISMAIL, A. GHAZAL, M. F. EL-ASMAR and A. A. ABDEL-RAHMAN
components were found in venoms from the scorpions Buthus minax, Buthus occitanus and Leiurus quinquestriatus. However, the bands found were not identical with those of A . amoreuxi venom, although cross-reactions occurred between some of the bands of B. mirtax, B. occitanus and L. quinquesiriatus . A cross-reaction was shown to occur between some bands of B. minax and L. quinquestriatus venoms using B. ntinax antivenin (EL-AS1alAx et al., 1973). REFERENCES BnLOZ.rr, L. (1971) Scorpionism in the Old World. In : Venomous Animals and Their Venoms, Vol. 111, Venomous Invertebrates, pp. 349-371, (Boer., W. and BUCKLEY, E., Eds.). New York : Academic Press. BROWN, G. L. (1938) The preparation of the tibialis anterior (cat) for close arterial igjection. J. Physiol., Load. 92, 22p. Bth.HRnvc, E. (1946) Observations on the isolated phrenic nerve diaphragm preparation of the rat. Br . J. Pharmac. Chemother. 1, 38 . E[rAs~taR, M. F., IsMnu, M. and OsMnN, O. H. (1973) Immunological studies of scorpion (Buthus minax, L. Koch) venom. Toxicon il, 9. GHA7~i , A., IsMan., M., ABDE>rRax~rr, A. A. and EtrAsMax, M. F. (1975) Pharmacological studies of scorpion (Androctonus amoreuxi, Aud. & Sav.) venom. Toxicon in press. GLENN, W. G., KEEanN, H. L. and WxrrreMOxe, F. W., JR . (1962) Intergeneric relationship among various scorpion venoms and antivenins. Science 135, 434. Istitnu, M., Osns.~N, O. H. and ErrAsMnR, M. F. (1973) Pharmacological studies of the venom from the scorpion Buthus minax (L . Koch). Toxicon 11, 15 . KoHrr, J. (1968) In : Chromatographic and Electrophoretic Techniques, Vol. Ii, Zone ElectrophoresLs, 2nd ed ., p. 84, ($MITFI, L, Ed .) . William Heinemann Medical Books Ltd. Oucx~rERt.oxr, O. (1948) In vitro method for testing the toxin producing capacity of diphtheria bacteria . Acta path. microbiol. Scand. 25, 186. PoTrER, J. M. and NoRZ~t, W. T. (1962) An immunological evaluation of scorpion venoms . J. trop . Med. Hyg, 11, 712.
TOXICON 1975 Vol. l3
IMMUNOLOGICAL STUDIES WITH SCORPION
(ANDROCTONUS AMOREUXI AUD. & SAV.) VENOM M.
ISMAIL, A. GHnzAL, M. F. EL-ASMAR * and A. A. AsnEh-RAxMAx Department of Pharmacology, Faculty of Pharmacy, University of Alexandria, Egypt (Accepted jor publication 14 May 1975) M. IsiHwlt., A. GI;"~"~, M. F. EL-AsMax and A. A. AHn~RexMerl. immunological studies with scorpion (Androctonus amoretexl Aud. & Sav.) venom. Toxlcon 13, 405-408, 1975 . An antiveninwasprepared forA. amoreuxi venom by hyper-immunizing rabbits. The antivenin protested mice against the lethal action of the venom and prevented blockade of twitch activity caused by the venom in both the phrenic nerve-hetnidiaphragm of the rat and the cat tibialis anterior preparation . The antivenin also prevented the cardiac stimulant and the uterine inhibitory effects of the venom . Using immunodiffusion and immuncelectrophoresis techniques, it was possible to reveal 7 prominentand 2faintprecipitin bandswith A. amoreuxi venom. Precipitin bands corresponding to individual venom fractions were also identified. Antigenic components were also found with venoms from the scorpions Bathos minax, Buthus occitaJUU and Lelurus quinquestriatus . The bands found, however, were not identical with those of A. amoreuxi venom. INTRODUCTION
Androctonus amoreuxi is widely distributed in Egypt and neighbouring countries (BALOZSr, 1971). No specific antivenin is available in Egypt against its venom despite the frequency of the scorpion stings . Individuals subjected to A. amoreuxi stings are usually treated with a poly antivenin prepared from scorpion venoms but not including A. atnoreuxi. BALOZET (1971), however, pointed out that "scorpion antivenins are rather specific". In a previous publication from this laboratory (GxAZa~l, et al., 1975), the venom from A. amoreuxi was found to possess potent neurotoxic and cardiotoxic properties. It was thought worthwhile, therefore, to attempt the preparation of a specific antivenin for A. amoreuxi and to test its protecting properties in experimental animals. TrIE SCORPION
MATERIALS AND METHODS
Venom was obtained from mature A. amoreuxl as described by Ist~,n, et al. (1973). Hyperimmunization of rabbits Male rabbits weighing between 2 and 2" 5 kg were kept in the animal house for 2 weeks before immunization . The rabbits were tested for natural immunity and only those whose serum showed no precipitin bands with A. amoreuxt venom were used. The rabbits were injected subcutaneously with A. amorercxivenom, each dose being emulsified in 0" 5 ml complete Freund's adjuvant (Difco Laboratories, Detroit, Mich .), according to the following schedule : two injections of 200 pg each in the first week, three injections of 300 Itg each in the second week and one igjection of 500 Etg in each of the fifth and the subsequent S weeks. Five days after the final dose, blood was withdrawn from tho marginal ear veins and the serum tested for positive precipitin bands. Rabbits showing positive precipitin bands were injected with 500 !ag venom and were bled 6 days later. The collected blood was left at 25 °C for 2 hr to form a spontaneous clot. Serum was obtained by centrifugation at 6000 rev per min, pooled, and thiomerosal (1 : 10,000) was added as a preservative. The serum was then distributed in small tubes and kept at -20°C until used. *Department of Biochemistry, Faculty of Medicine, Ain Shams University, Cairo, Egypt. TOXJCON 1975 V°t. J3
404
406
M. ISMAIL, A. GHAZAL, M . F. EL-ASMAR and A. A. ABDEL-RAHMAN
Immuirochemkal studies Electrophoresis of the crude venom was carried out as described by Gxnznr. et al. (197 . Fractions separated by cellulose acetate electrophoresis were eluted with 0'3 ~ NaCI solution and adjusted to a final concentration of 1 mg per ml . Immunodiffusion experiments were carried out on lantern slides (5 x 5 cm) using 1'2~ Noble agar (Difco Lab., Detroit, Mich.) in 0~9~ NaCI solution. Sodium azide in a concentration of 005 ~ was added to retard bacterial growth. Double diffusion experiments were carried out as described by OUCFITERLONY (1948). The wells were filled with 20 ul volumes of either the crude venom, the various fractions separated by cellulose acetate electrophoresis or the antiserum. After developing of the precipitin bands (48 hr at 25°C), the plates were washed for 24 hr in saline, dried and stained with amidoschwartt 10 B (0~5 ~ in 5 acetic acid) for 7 min, washed with methanol : acetic acid (9 : 1), dried in air and photographed . Immunoelectrophoresis was carried out by the transfer method (KoxN, 1968) using lantern slides (5 x 20 cm) covered with agar. A pattern indicating the desired relative positions of the electrophoretic and antivenom strips was drawn on white paper. This pattern was clearly visible through the slide when it was placed underneath . The electrophoresic run of the venom (50 ul samples of a 20 mg per ml solution) was carried out on cellulose acetate strips (50 x 200 mm) using barbitone buffer, 005 ionic strength, pH 8~6 for 150 min and applying 25 V per cm . At the end of the run, the strips were cut longitudinally into three equal parts. The middle third was dried in hot air and stained with Ponceau S (0'2 % in 3 ~ trichloroacetic acid) and used to locate the protein bands on the other two unstained thirds. Afilter paper strip (Whatman No . 3, approximately 2 mm wide) was impregnated with the antivenin (80 pl) and placed on the gel at the previously indicated position . One third of thecellulose acetate strip was placed at one side of the antivenom strip. The bands on the remaining third were cut out and placed, each 0~5 cm apart, at the other side of the antivenin strip. The plates were then placed in a moist chamber at room temperature (25°C) and diffusion was carried out for 248 hr. The slides were then washed in 0~9~ NaCI solution, dried and stained with Ponceau S, washed with 5 ~ acetic acid, dried in air and then photographed . Neutralization tests In these experiments A. amoreuxi venom was incubated with the specific antivenin for 1 hr at 37°C in the proportion of 1 mg venom to 4 ml serum. (a) Protection against venom lethality in mice . The venom-antivenin mixture was shaken and a dose equal to the tn bo (088 mg per kB, G><;az~L et al., 1975) was injected intramuscularly into each of 10 mice. Doses of the incubated mixture equal to 2-5 times the ~su were injected each into a group of 10 mice . All mice were observed for 24 hr after injection. (b) Protection against thepharmacological effects of the venonr. In these experiments doses of the venomantivenin incubated mixture equivalent to doses ofthevenomshown to producepronounced pharmacological effects were tested on the following preparations : the isolated phrenic nerve"-hemidiaphragm of the rat (BÜLHRING, 1946), the cat tibialis anterior muscle (Bxowx, 1938), the isolated rabbit and guinea pig hearts (LangendorlF preparation) and the spontaneously contracting rat uterus in Ringer-Locke solution. RESULTS
In the immunodiffusion experiments precipitin bands were obtained with venom concentrations as low as 50 ltg per ml. However, in most of the experiments in both the immunodiffusion and the immunoelectrophoresis, a venom concentration of 20 mg per ml was used. Six prominent and one faint precipitin bands were shown with crude A. amoreuxi venom (Fig. 1) using the immunodiffusion technique. In some experiments, some of the bands were fused together. Precipitin bands were also revealed with the first four venom fractions closest to the origin . Thus each of fractions 1 and 2 revealed one precipitin band and each of fractions 3 and 4 showed three precipitin bands (Fig. 1). Testing the eluates of the areas between the different venom fractions showed a precipitin band to be revealed only by the area between fractions 3 and 4. This band cross-reacts with one of the precipitin bands formed by fraction 3. In the transfer immuncelectrophoresis technique five prominent precipitin bands were shown, while in the cut-transfer immunoelectrophoresis 7 prominent and 2 faint precipitin bands were revealed (Fig. 2). In contrast to the immunodiffusion technique precipitin bands, corresponding to the fraction at the origin, were found in the transfer immunoTOXICON 1975 Vol. 13
FIG . I . DOUBLE DIFFUSION REACTION OF RABBIT SERUM ANTIVENIN FOR A . a1Y.ore1(Xi VENOM (CENTRAL WELL) . V, A . amoreuxi venom (20 mg per ml); Or, band at the origin (1 mg per ml) ; F,~ ( l mg per ml),
the first 5 fractions in sequence from the origin ; separated by cellulose acetate electrophoresis . FIG . 2. IMMUNOELECTROPHORETIC PATTERN OF RABBIT SERUM VENOM (IN TROUGH).
ANTIVENIN FCR A . ali:Ofel(Xl
Or, band at the origin (1 mg per ml); FIB (1 mg per ml), the first 5 fractions in sequence from the origin ; separated by cellulose acetate electrophoresis. Upper trace, 'cut-transfer' immuncelectrophoresis; lower trace, transfer immuncelectrophoresis . Experimental conditions as in text . FIG . 3 . DOUBLE DIFFUSION REACTION OF RABBIT SERUM ANTIVENIN FOR A . QIY.'orEGXi VENOM (CENTRAL WELL) WITH OTHER SCORPION VENONS . A, A . atroreuxi venom ; Bm, Buthus minax venom ; Bo, Buthus occitanus venom ; Lq, Leiurus
yuirryuestriatus venom. Venom concentration 20 mg per ml .
TOX/CON 1975 Vol. l3
f.p . 406
FIG. 4. EFFECT OF INCUBATING A. pIY.O/PGXl VENOM WITH RABBIT SERUM ANTIVENIN ON THE RESPONSE OF THE RAT PHRENIC NERVE-HEMIDIAPHRAGM PREPARATION.
Rat phrenic nerve-hemidiaphragms in Krebs-Henseleit solution stimulated at 0~1 Hz . Bath capacity 30 ml . (a) : ", A. amoreuxt venom (300 ug). (b) : ",rabbit serum antivenin (12 ml); ", A . amoreuri venom (300 llg) -i rabbit serum antivenin (1 ~2 ml).
TOXlCON J975 Vol. !?
FIG, S . EFFECT OF INCUBATING A. ü,')IOICüSl VENOM WITH RABBIT SERUM ANTIVENIN ON THE RESPONSE OF THE CAT TIBIALIS ANTERIOR MUSCLE PREPARATION.
Male cat (4 kg) under chloralose anaesthesia. Maximal twitches of the tibialis anterior muscle elicited by stimulation of the nerve once every 10 sec. ", Venom (250 ug)r antivenin (1 ml); O, venom (250 ug). Injections were given close-arterially.
FIG. G. EFFECT OF INCUBATING A, arifOIEüX! VENOM WITH RABBIT SERUM ANTIVENIN ON THE RESPONSE OF THE SPONTANEOUSLY CONTRACTING RAT UTERUS .
Uterus in natural oestrus. Bath capacity 30 ml . ", Venom (60 ug); ", rabbit serum antivenin (0'3 ml) ; O, venom (60 Ilg) + rabbit serum antivenin (0~3 ml) ; W, wash. Contractions magnified 4 times under a constant load of 2 g using an auxotonic lever. TOXICON 1975 Vol. J3
Scorpion Venom Antivenin
407
electrophoresis (Fig. 2). In addition a precipitin band corresponding to fraction 5 was shown in the cut-transfer immuncelectrophoresis technique (Fig. 2). Prominent precipitin bands were also shown with venoms from Bathos minax (2 bands), Bathos occitam~s (3 bands) and Leiurus quinquestriatus (3 bands) using the immunodiffusion technique and venom concentration of 20 mg per ml (Fig. 3). The bands, however, were not identical with those of A . amoreuxi venom (Fig. 3). Incubation of A, amoreuxi venom with the specific antivenin protected the mice against the lethal action of a dose of the venom equivalent to double the LDsu. It also protected 80 and 60 % of the mice against the lethal action of doses of the venom equivalent to 4 and 5 times the I,n b n dose, respectively . The antivenin markedly attenuated the increase in twitch height of the rat phrenic nerve-hemidiaphragm by the venom (Fig. 4) and prevented the venom-induced contracture of the cat tibialis anterior muscle (Fig. ~. In both preparations the subsequent blockade of twitch activity was abolished. The antivenin also abolished the uterine inhibitory effect of the venom on the spontaneously contracting rat uterus (Fig. 6), and the initial negative isotropic and chronotropic effects and the subsequent marked positive isotropic effect on the isolated rabbit and guinea pig hearts . DISCUSSION
Fractionation and pharmacological investigations of A. amoreuxl venom were carried out by Gxnzni, et al. (197 . The venom was shown to possess potent neurotoxic and cardiotoxic properties and was fractionated by cellulose acetate electrophoresis at pH 8~6 into 8 cathodic fractions. Lethality and pharmacological activities were associated with only 3 venom fractions. Of these fractions, fraction 4 was the only lethal fraction and in addition it caused cardiac stimulation, uterine relaxation and an increase in the twitch height of the rat phrenic nerve-hemidiaphragm. Fraction 3 caused uterine relaxation and increased twitch height of the rat phrenic nerve-hemidiaphragm. Fraction 5 was only active on the cat tibialis anterior muscle causing contracture followed by blockade of twitch activity. The other fractions were without effect . In the present study the antivenin prepared against A . amoreuxi venom by hyperimmunizing rabbits protected the mice against the lethal action ofthe venom. This effect could be explained by the ability of the antivenin to neutralize the effects of fraction 4, the only lethal fraction. The formation of precipitin bands against fractions 3-5 could explain the protecting activity of the antivenin against the uterine inhibitory, the cardiac stimulant and the contracture and blockade of twitch activity of muscle preparations produced by the venom. The formation of more than one precipitin band with fraction 4 indicates that this fraction is probably composed of more than one protein. Attempts are being made to further purify this fraction . The use of `cut-transfer' immunoelectrophoresis, in which the individual fractions are cut out and placed, each 0~5 cm apart, on the side of the antivenin strip, offered better differentiation and identification of the precipitin bands than with the ordinary immunoelectrophoresis or immunodiffusion techniques . In addition, precipitin bands corresponding to the individual fractions could be recognized and used in explainingthe protecting activities of the antivenin. GLEnx et al. (1962) have investigated the intergeneric relationships of venoms obtained from scorpions separated by geographical barriers and showed that the venoms contained certain antigenic components in common. Also, Parrm and Nox~t (1962) showed that a large number of antigenic components are shared by members of the families Buthidae and Vejovidae . Using the immunodiffusion technique and A . amoreuxi antivenin, antigenic TOXICON 1975 Vol. l3
408
M. ISMAIL, A. GHAZAL, M. F. EL-ASMAR and A. A. ABDEL-RAHMAN
components were found in venoms from the scorpions Buthus minax, Buthus occitanus and Leiurus quinquestriatus. However, the bands found were not identical with those of A . amoreuxi venom, although cross-reactions occurred between some of the bands of B. mirtax, B. occitanus and L. quinquesiriatus . A cross-reaction was shown to occur between some bands of B. minax and L. quinquestriatus venoms using B. ntinax antivenin (EL-AS1alAx et al., 1973). REFERENCES BnLOZ.rr, L. (1971) Scorpionism in the Old World. In : Venomous Animals and Their Venoms, Vol. 111, Venomous Invertebrates, pp. 349-371, (Boer., W. and BUCKLEY, E., Eds.). New York : Academic Press. BROWN, G. L. (1938) The preparation of the tibialis anterior (cat) for close arterial igjection. J. Physiol., Load. 92, 22p. Bth.HRnvc, E. (1946) Observations on the isolated phrenic nerve diaphragm preparation of the rat. Br . J. Pharmac. Chemother. 1, 38 . E[rAs~taR, M. F., IsMnu, M. and OsMnN, O. H. (1973) Immunological studies of scorpion (Buthus minax, L. Koch) venom. Toxicon il, 9. GHA7~i , A., IsMan., M., ABDE>rRax~rr, A. A. and EtrAsMax, M. F. (1975) Pharmacological studies of scorpion (Androctonus amoreuxi, Aud. & Sav.) venom. Toxicon in press. GLENN, W. G., KEEanN, H. L. and WxrrreMOxe, F. W., JR . (1962) Intergeneric relationship among various scorpion venoms and antivenins. Science 135, 434. Istitnu, M., Osns.~N, O. H. and ErrAsMnR, M. F. (1973) Pharmacological studies of the venom from the scorpion Buthus minax (L . Koch). Toxicon 11, 15 . KoHrr, J. (1968) In : Chromatographic and Electrophoretic Techniques, Vol. Ii, Zone ElectrophoresLs, 2nd ed ., p. 84, ($MITFI, L, Ed .) . William Heinemann Medical Books Ltd. Oucx~rERt.oxr, O. (1948) In vitro method for testing the toxin producing capacity of diphtheria bacteria . Acta path. microbiol. Scand. 25, 186. PoTrER, J. M. and NoRZ~t, W. T. (1962) An immunological evaluation of scorpion venoms . J. trop . Med. Hyg, 11, 712.
TOXICON 1975 Vol. l3