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>
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>
