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Nov 20, 1978 - however limited by the risk that the patient might develop such allergic reactions as ana- phylactic shock, serum sickness, Arthus reaction, etc.
Toxfrnn, Vol. 17, yy . 326-330. ~t,l PerQamon Press Ltd. 1979. Printed in Great Britain.

0041-0101/79/0501-0326502.00/0

PURIFICATION OF MONOSPECIFIC ANTISERA AGAINST THE VENOM OF THE CAPE COBRA (NAJA NIVEA) THOMAS MADSEN, HENRIK LUNDSTRÖM

and

.TAN FOHLMAN

Institute of Biochemistry, University of Uppsala, Box 576, 5-751 23 Uppsala, Sweden (Accepted jor publication 20

November 1978)

cause-directed snake envenomation therapy is immunological. Serotherapy is however limited by the risk that the patient might develop such allergic reactions as anaphylactic shock, serum sickness, Arthus reaction, etc. To circumvent this, pepsin-digested antibodies have been fractionated by ammonium sulphate precipitation to yield (Fab') Q fragments which are less allergenic than intact immunoglobulins (HARMS, 1948 ; GRASSET and CHRISTENSEN, 1947) . Tn this communication we have used horse (Fab') $ fragments for the isolation of monospecific anti-neurotoxins by affinity chromatography . Naja nivea venom was fractionated by an initial gel filtration on Sephadex G-75 to give non-toxic higher molecular-weight (mol. wt .) protein fractions, a neurotoxic fraction in the size range 7-8000 daltons and low mol. wt nucleosides. The neurotoxic fraction was further separated by ion exchange chromatography on Bio-Rex 70 at pH 6~5, essentially as described by KARLSSON et al. (1971) . As evidenced by amino acid analysis five homogeneous proteins were isolated after these two steps. A further ion-exchange chromatography on Sulfopropyl-Sephadex C-25 at pH 5~5 using a linear concentration gradient of 0~5 vs 1 M ammonium acetate yielded two additional pure components . Two affinity gels were prepared by coupling to Sepharose 4B with the CNBr method (AxÉrr et al., 1967) . Two of the isolated N. nivea neurotoxins were used for coupling, along with a short neurotoxin from the venom of the common sea snake (Enhydrina schistosa) (KARLS .SON et al., 1972). We thus prepared one gel liganded with a long type neurotoxxn (71 amino acids, 5 disulfide bonds) and one gel liganded with a toxin of the short type (60 amino acid residues, 4 disulfide bridges) (KARLSSON, 1978). The affinity gels were packed in small columns equilibrated with 0~5 M NaCI in Na HCO s-buffer, pH 8~3 . Crude pepsin-digested antisera (kindly prepared for research purposes by Dr. CHRISTENSEN) were applied and non-adsorbed proteins were washed out with 1 M NaCI. Desorption was achieved using 0~2 M glycine-HCI, pH 2~8 . The antibodies were pooled and dialyzed against 0~1 M ammonium acetate at 4°C for 24 hr and lyophilized. Estimation of concentration was done spectrophotometrically assuming A'~~ = 1~5. Estimates of neutralizing capacity were made by mouse assays at three different dose levels, 2 X LDaO, 5 x LD50 and 10 x LDsu. Venom and antibodies were preincubated together for 45 min at room temperature before i.v. injection. Some assays were done s.c. with little difference. Four mice (20-25 g) were used at each dose level. Dosages of crude venom were based on weight . The fractionation of the crude venom is shown in the flow scheme, Fig. 1 . Used in the coupling experiments were the delineated fractions, i.e., the most retarded Bio-Rez 70 THE oNLY

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neurotoxin and the toxin a (Bores et al ., 1971). We could not demonstrate the toxins ß anal although the most retarded Bio-Rex fraction is similar to ß (Table 1) . Two other neuro-

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toxins and two cardiotoxins, including a non-toxic variant, analyze as homogeneous proteins and the compositions agree completely with toxin a (BoTFS et al., 1971) and toxin VIII and VII2 (BOTES and Vu.rosrr, 1976) although Vn2 is non-toxic in our hands (Table 1). We do not know whether the dissimilarities are due to the different origin of the venom, possibly reflecting genetic variation, or are consequences of the different separation techniques used. N. nivea occurs in a rather small geographical region but with much climatic variation and certainly colour variations are frequent ranging from white, yellow, black and speckled . The purpose of this paper was not to purify N. nivea venom, but to prepare neurotoxins for purification of antiserum. Therefore this question was not pursued in detail . Another important factor may be that we only used 500 mg crude venom, whereas BOTES and VuaoErr (1970) used 9 g and thus intrinsically have 20 times higher sensitivity. The neurotoxins used for the affinity chromatography step constituted 7~8 ~ of total venom. Coupling yields to the Sepharose 4B were 95~ and the total recovery of antibody was around 97 ~. Toxin a gives rise to the most antibodies (63~ of the isolated antineurotoxins), probably because it is a better immunogen, since dose-response in the production of immunoglobulins is not linear . The potent antibodies constitute 8 ~ of the crude preparation (92~ apparently being non-antineurotoxins). Some material (less than 1 ~) remains on the columns which might represent denatured protein or antibodies with the highest affinities. We have obtained the same rn6s-value, 450 ltg/kg, for the crude venom as was reported earlier (BOTES and STRYDOM, 1971). The neutralizing capacities of the purified antisera vs the crude preparation is shown in Table 2. If only anti-a is used, about two l n6o of the crude TAHCE 2. NEUTRALIZING} CAPACITY OF PURIFIED (Fab~, Venom dose

mg ""crude"', (Fab'),/kg

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27 89 233

mg Purified (Fab'),t 2"4 12 30

" Crude (Fab~, is the starting material . to combination of Nq/a nivea anti-toxinsand F .nlrydrüra schtstosa anti-toxins in the same ratio the toxins occur in NgJa nivea venom, i.e . 63 ~ long type and 38 ~ short type. za of the crudevenom was 4501Ig/kg.

venom canbetolerated with survival . The combined anti-short type antisera could notneutralize two LD 6 p of crude venom. Two mice were given 600 mg/kg nonadsorbed antibodies, but this could not neutralize 10 r n60. However, the combination of anti-short and anti-long type neurotoxin antibodies in the 3:5 ratio at which these types oftoxins occur in the venom had about 8 times the neutralizing capacity toward crude venom than did the whole Antisera . In a severe bite the dose of foreign protein could thus be reduced from About 100 ml antivenom to about 10 ml, which would drastically relieve the burden on the patient's allergic response . Also, the speed of injection and ease of operation are important factors . If a 5 solution is made and a normal bite is assumed to represent 1-2 c.n6o, 5 ml ought to be a sufficient dose for counteracting the ill effects of the venom. This should also reduce the ill effects of the antiserum. It is common clinical experience that the severeness of the adverse allergic reactions) is dose-dependent (FoucwRn, personal communication). Certain elapid venoms, like those of the mambas and cobras, ought to be ideally suited for this type of antiserum refinement . These venoms contain long and short neurotoxins usually totalling less than 30~ of the protein (KAxissox, 1978). Exceptions are, for instance, the

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common sea snake (Enhydrina schistosa) which has about 70~ neurotoxins and about 10~ myotoxins (Fol-u.1KAx and EAxsR, 1977) and the South American rattlesnake (Crotalus durissus terrifi'etts) which can have up to 90 ~ crotoxin. The advantage with the latter venoms is that no purification before immobilization of the venom should be necessary. We have observed that i.v. injection of venom is difficult to reverse even with an i.v. injection of antisera within 5 min. This probably reflects the fact that the antigen antibody reaction is in this case more sluggish than is the binding of the toxins to the acetylcholine receptor. The same observation holds also for presynaptic snake venoms, and emphasizes the need for speedy antisera administration (RAMLAU et al., 1978). In a natural bite s.c. deposition of venom is much more likely, thus allowing the circulating antibodies more time to detoxify the venom as it enters the bloodstream. Acknowledgements-We thank Dr. P. A. Ctntrsi$xseN for generous gift of N. nivea venom and antiserum and Dr . D. E~see for E. schlstosa neurotoxin and aid with amino acid analysis . RRFRRRNCES Ax~rr, R., PORATH, J. and Eaxs~cx, S. (1967) Chemical coupling of peptides and proteins to polysaccharides by means of cyanogea halides. Natrue (Land.) 214, 1302 . Bats, D. P. and Vn tosrr, C. C. (1976) The amino acid sequence of three non~urarimimetic toxins from N4ja nlvea venom. Blochim. biophys. Acto 446, 1. Booms, D. P., S~rxYnoM, D. J., AxnaxsoN, C. G. and CmusrExssx, P. A. (1971) Snake venom toxins : Purification and properties of three toxins from Naja nlvea (cspo cobra) venom and the amino acid sequence of toxin S. J. biol. Chem. 246, 3132 . FO v, 7. and E~x~x, D. (1977) Isolation and characterization of a lethal myotoxic phospholipase A from the venom of the common sea snake (i:nhyarlna schlstosa) causing myoglobinuria in mice. Toxlcon 13, 385. Fot~.u~x, J. and EAKER, D. (1977) Isolation and characterization of a lethal mytoxic phospholipase A from the venom of the common sea snake (Enhydrina schlstosa) causing myoglobinuria in mice . Toxtcon 16, 385. Gs~r, E. and CxntsreNSex, P. A . (1947) Enzyme-purification of polyvalent antivenin against southern and equatorial African colubrine and viperine venoms . 7rans. R. Soc. Trop. med. Hyg. 41, 207. Hex~ts, A. J. (1948) The purification of antitoxic plasmas by enzyme treatment and heat denaturation . Biochem. J. 42, 390. Kaxtssox, E., AxNeaea, H. and EAKER, D. (1971) Isolation of the principal neurotoxins of two Naja raja subspecies . Eur. J. Blochem. 21, 1. K~tussox, E., Enx~t, D., FaYxi.urm, L. and Kwncx, S. (1972) Chromatographic separation of F.nhydrina schistosa (common sea snake) venom and the characterization of two principal neurotoxins . Btochemlstry 11, 4628 . Kaxcssorr, E. (1978) In : Handbook ofExperimental Pharmacology, Vol. 52 (Lse, C. Y., Ed .) . Berlin : Springer, in press. Rw~u, J., BocK, E. and Foru.ntwx, J. (1979) Production of antibodies against modified taipoxin and immunachemical analysis of the subunits a, ß and 7. Toxlcon 17, 43 .