- Email: [email protected]
Enzyme-linked Immunosorbent Assay (ELISA) in assessing antivenom potency
Toxkon, Vol. 17, pp. SI1-S15 . ® Pergamon Press Ltd . 1979 . Printed in Great Britain.
0041-0101/79/0901-031IS02.00/0
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) IN ASSESSING ANTIVENOM POTENCY R. D. G. TIiEAKSi'ON and H. A. Re1n
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, United Kingdom (Accepted for publication 5 March 1979) ABSTRACT R. D. G. THFAK31'O1V and H. A. Rte. Enzyme-linked immunosorbent assay (ELISA) in assessing antivenom potency. Toxicon 17, 511-515, 1979.-Enzyme-linked immunosorbent assay (ELLSA) has been investigated as a screen of antivenom potency. One batch from each of three commercial sotuces and from two rabbit research antivenoms were established as the five group reference antivenoms and the median effective doses (En bos) for neutralising four venoms medically important in Africa were estimated in mice. Reference curves were prepared relating EDbo s to optical densities with ELISA . The En 6os of 38 different batches of test antivenom were estimated by ELISA, and ELISA eo, os were then compared with in vivo mouse ensos. Excellent correlation was obtained . The method is rapid, cheap, simple, economic in amounts of venom used, and most importantly, greatly reduces the need for live animals. If validated in an extended range of venoms and antivenoms, ELISA screening of antivenom potency should be a useful standard procedure. TNTRODUCTION
assays of biological substances such as antivenoms, it is highly desirable to avoid suffering and death ofanimals by substituting in vitro methods, provided such methods measure life-saving capability with precision similar to that of in vivo methods. liut in vitro methods so far tried have not been successful (CHRISTENSEN, 1968) ; we have therefore investigated enzyme-linked immunosorbent assay (ELISA) in this problem (THEAKSTON et al., 1977). Irr PorErrcY
MATERIALS AND METHODS Venoms and antivenoms Pooled representative samples of four venoms medically important in Africa were obtained by personally milking adult specimens of the puff adder (Batas arietans), carpet viper (Echis carinatus), Egyptian cobra (Naja haje) and spitting cobra (Naja aigricollis). All the snakes were maintained in our herpetarium and were kindly supplied from north Nigeria by Drs. R. N. H. Pugh and D. A. Walrell. Venom was weighed, freeze-dried and stored in the dark at 4°C. Monospecific antisera were raised in rabbits against B. arietans and E. carinatus venoms (Txsnxsrorr et al ., 1977). Relevant commercial therapeutic antivenoms (Tables 1 and 2) were from Behringwerke AG (Behr), Postachliessfach 167, West Germany (kindly donated) ; the South African Institute for Medical Research (SAIMR), P.O . Boz 1038, Johannesburg ; and the Haffkine Institute (HTj, Parel, Bombay l2, India. Behr and SAIMR antivenoms are fluid; HI antivenom is freezedried. Outdated antivenoms from these throe institutes were kindly donated by the Zoological Society of London. All batches of antivenom were kept in the dark at 4°C until use. The investigations reported in this paper were performed during April-November 1978 . Lethal toxicity of venoms
Lethal toxicity was assessed by subcutaneous (s .c .) injection of venom in 02 ml physiological saline into the hind thigh of mice weighing 18-20 g. Six mice were used at each venom dose . The median lethal dose (tn, o) was calculated by probat analysis (Fnvtv>:v, 1971) of deaths occurring within 24 hr of venom injection. Lobos and 95 ~fiducial limits in parentheses were 1701tg/mouse (140-210) for B. arietansvenom,120 ug/mouse (84-150) for E. carirtatus venom, 14 ug/mouse (13-16) for N. haje venom and 106 ug/mouse (80-120) for N. rrigrtcollis venom.
51 2
R. D. G. THEAICSTON and H. A. REID
Estlrnation ojaruivenam neutralising ED bo F1Ve LD6oS of venom in 0~2 ml of physiological saline was injected s.c . into mice and then varying amounts of antivenoms in 7 graded doses (200~~25 itl) made up to a total of 0~2 ml in physiological saline, were injected rapidly into the tail vein of mice (6 mice per dose). After observing the mice for 24 hr, the neutralising median effective dose (®bu) and 95 ~ fiducial limits were calculated by probit analysis. The ®bn is the least amount of antivenom needed to prevent death of half the mice in a group (thus, the lower EDbu, the higher the neutralising ability) . If 200 ul antivenom failed to save at least half the mice, the antivenom was judged ineffective. Group reference antivenoms One batch of antivenom from each of the three institutes and from a pooled batch of the two monospecific rabbit antivenoms were arbitrarily selected as Group Reference and ED bos against each of the four venoms were determined . A range of multiples and fractions of these EDbu s, from 6 times the EDbp down to 1/64th EDba, were diluted 1/50 (commercial antivenoms) and I/1000 (rabbit antivenoms) in PBS "Tween" and assayed for each of the four relevant antibodies using ELISA (Ti1EAICSTON et al., 1977). The crude rabbit antivenoms were more diluted than the refined commercial antivenoms because the former give a much higher optical density with ELISA than the latter at a given ED bo . Commercial antivenoms were incubated with substrate for 40 min, rabbit antivenoms for 20 min. Table 1 shows the ED6o of each reference antivenom and its corresponding optical density with ELLSA. All assays were in duplicate. Group reference curves of optical densities from ELISA were then plotted against the mouse in vtvo ED6 u for each of the four venoms . TABLE l . NEUTRALISING ED6 pS AND ELISA READINGS Antivenom Source, batch No. Behringwerke 317 SAIMR 0938 Haffkine 135-E Rabbit R6 Rabbit R5
Labelled active against" BAV ECV NHV NNgV BAV ECV NHV NNgV ECV (N. naja)
Expiry date (month/yr) 8/77 7/75 10/74
Venom tested BAV ECV NHV NNgV BAV ECV NHV NNgV ECV NHV NNgV BAV ECV
OF
REPERENCE ANTIVENOMS eDba (lil/mouse)t 64 ( 29-121) 34 ( 15- 66) 192 (100-350) 182 ( 95-340) 22 ( 8- 46) 16 ( 6- 36) 35 ( 16- 68) 48 ( 22- 89) 50 ( 22- 92) 144 ( 75-230) 161 ( 85-299) 35 ( 16- 72) 64 ( 29-121)
ELISA optical density at 400 nm 0700 0520 0295 0l42 0206 0335 0l36 0089 0530 0256 0l37 098l 1012
'Venoms : BAV = Bais arletans ; ECV = Echis carinates ; NHV = Naja haje ; NNgV = Naja nigricolhs . tNumbers in parentheses are 95 ~ fiducial limits . Investigation of unknown antivenoms By comparing (spectrophotometrically) the optical densities of different doses of unknown antivenoms (200-3125 p1 diluted 1/50 in PBS "Tween") with those of the relevant reference curve at 1 ED 6o, the theoretical ELISA ED 6, was obtained . The same conditions applied with crude rabbit antivenoms except that 1/1000 dilutions were used . In vivo ED bos were then determined in mice, using 7 graded doses ranging from 200 to 625 IIl with 6 mice at each dose . The results were compared with ELISA EDb,s and optical density/ED6 u curves were collated with relevant reference curves . RESULTS
The close correlation between the ELISA ED bo and mouse in vivo ED 6o is shown in Fig . 1 and in Table 2. For 16 E. carinatus venom rabbit antivenoms the correlation coefficient was 097 (P < 0001) ; similar results were obtained with 10 BAV rabbit antivenoms. With the 12 test batches of commercial antivenoms against E. carinatus venom, correlation was again highly significant (r = 092 ; P < 0001). ELISA optical density/mouse ED6o curves of test antivenoms and their appropriate reference curves were very similar ; Behr and HI curves were almost identical whereas SAIMR gave lower optical densities and crude rabbit antivenom showed a much more intense colour reaction.
51 3
ELLSA and Antivenom Potency
R ioo
0 W
In vivo eo yo , wl
Z(~Î
FiO. 1. CORRELATION BETWEEN NEIJTRALL41N0 ED S p FROM ELISA AND MOUSE In VIVO ED~p (r = 0'96 ; P < U~001) .
DISCUSSION
Most ofthe commercial antivenoms investigated were `outdated' atthe time we tested them and the neutralising potency found in our tests confirms the conclusion by CHRISTENSBN (1975) that it is unrealistic and wasteful to limit the `life' of refrigerated refined antisera to 5 yr. Christensen also noted that opacities developed before potency was lost. All the antivenoms we tested were clear, except SAIMR batch 1323 (which nevertheless retained significant neutralising ability despite having opacities) . In general, SAIMR antivenoms showed the most potent neutralising ability and this has been confirmed clinically for E. carinatus venom in man (WARRELG Ct aL, 1974). The ability of SAIMR antivenom 1879, labelled as active only against E. carinatus venom to neutralise other venoms can be explained by previous immunisation ofthe horses concerned with other venoms (CHRISTENSEN, personal communication, 1978). The poor neutralising ability of Echis antivenom against E. carinatus venom from different geographic areas has also previously been observed both clinically (WARRELL and ARNETT, 1976) and in the laboratory (LATIFI, 1973). ELISA measures antibody (or antigen) and we realise that antigen-antibody reactions of venoms and their degree of toxicity are two distinct and sometimes unrelated things (SCHÖTTLER, 1951). Antigens may be nontoxic and toxins may be non-antigenic. Nevertheless, our preliminary studies suggest sufficient correlation between the in vivo mouse EDao and the ELISA optical density to warrant further exploration of ELISA as a simple in vitro screening assay, both ofcommercial therapeutic antivenoms and of experimental antisera . At present the ELISA reference standard has to be separately established for each commercial group or each source . This may be due to different methods of production or purification ; certainly crude antivenoms give a much higher optical density with ELISA at a given En so . We can screen 5 antivenoms from one commercial group on one ELISA plate by simply placing on the plate the known group reference at 1 x aD6o and different dilutions of the 5 test antivenoms . The point of colour match (optical density) between the reference antivenoms and the test dilution, enables the quick calculation ofthe Ens o of the test antivenom. Compared with in vivo potency tests, ELISA is simpler (if a spectrophotometer is not
51 4
R. D. G. THEAKSTON and H. A. REID TABLE
2.
NEUTRALISINQ ED6pS
Antivenom Labelled active Source, batch no. against" Behringwerke BAV 1472 NHV NNgV Behringwerke 1497 Behringwerke 1527 Behringwerke 1575 Behringwerke 1576
BAV ECV NHV NNgV BAV ECV NHV NNgV ECV NHV
(ELISA
Expiry date (month/yr) 2/72 1/73 12/73 8/75
10/81
SAIMR 1323
BAV ECV NHV NNgV BAV ECV NHV NNgV BAV Naja sp.
SAIMR 1773
BAV Naja sp .
12/75
SAIMR 1879
ECV
5/80
SAIMR 2325
BAV Naja sp .
6/80
Haffkine 420-D
ECV (N. naja)
2/81
Haffkine 446-G
ECV (N. naja)
8/81
Behringwerke 207018
AND In VIVO MOUSE) OF TEST ANTIVENOMS
9/75
10/72
Venom tested" BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV
NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV ECV NHV NNgV ECV NHV NNgV
ELLSA Eubo
(1tl/mouse) 46 47 175 122 nil 75 110 60 57 23 117 nil 97 46 l25 nil 67 48 107 nil 23 22 200 100 47 90 25 142 23 200 40 50 50 9 21 110 21 30 30 67 100 nil nil 100 nil nil
//1 l'il'O EDSp
(ul/mouse)fi 40 ( l9- 73) 60 ( 35- 98) 200 (140-350) l19 ( 52-230) nil 70 ( 32--138) 122 ( 46-356) 100 ( 54-180) 63 ( 27-109) 28 ( 13-- 56) 160 ( 88-350) nil 90 ( 38-145) 49 ( 22- 89) 13G ( 72-270) nil 68 ( 31-115) 55 ( 25- 96) 200 (120-360) nil 22 ( 10- 45) 32 (15- 64) nil 137 (104-199) SO ( 24- 99) 74 ( 36-120) 28 ( 13- 56) 150 ( 78-260) 28 ( 12- 57) 160 ( 84-290) 35 ( 15- 66) 49 ( 23- 87) 25 ( l2- 54) l3 ( 4-- 32) 75 ( 39-140) 150 ( 80-290) 20 ( 9- 42) 40 (19- 73) 31 ( 14- 59) 69 ( 31-113) 87 ( 39-165) nil nil 83 ( 36-134) nil nil
"Venoms : BAV = Bltis arietans ; ECV = Echls carlnatus; NHV = Naja haje ; NNgV = Naja nigricollis . tNumbers in parentheses are 95 ~ fiducial limits .
available, antivenoms can be assayed with experience to the equivalent of 0050 optical density by visual matching) and more rapid . The economy in venom and, more importantly, in animals is notable . We are currently extending ELISA screening of antivenom potency to other venoms and antivenoms with a view to validating the technique as a standard procedure . Acknowledgements-We thank Miss S. B. J.
MACFARLANE
for advice on statistics, Mrs. D.
InnoN
and Miss A.
ELLSA and Antivenom Potency
S15
RItNMER for technical help, Mrs. HEATHER HUXLEY for secretarial work, and the Ministry of Overseas Development and the Medical Research Council for financial support. REFERENCES CHRLSTENSEN, P. A. (1968) The venoms of central and south African snakes . In : Venomous Animals and Their Venoms, Vol . 1, Ch . 16, p. 437, (BUCHERL, W., BUCKLEY, E. and DELOFEU, V. Eds.) . New York : Academic Press. C.HRI3TENSEN, P. A. (1975) The stability of refined antivenin. Toxlcon 13, 75 . FINNEY, D. J. (1971) Probit Analysis, 3rd edition. Cambridge. LArtrt, M. (1973) Studies of the venom of Iranian Echis carinatus in comparison with those of Pakistan and Eritria . Proc. 9th lnt. Cong. Tropical Medicine and Malaria, Athens, 14-21 October. Abstract 107. SCHÖITLER, W. H . A. (1951) Antigen-antibody relations in the present antivenin production of Brazil .
Am . J. trop . Med. 31, 500 .
THEAKSiON, R. D. G., LLOYD-JONES, M. J. and REID, H. A. (1977) Micro-ELISA for detecting and assaying snake venom and venom-antibody. Lancet ü, 639. WARRELL, D. A., DAVIDSON, N.MCD ., ORMEROD, L. D., POPE, H. M., WATKINS, H. M., GREENWOOD, B. M. and REID, H. A. (1974) Bites by the saw-scaled or carpet viper (Echis carinatus) : trial of two specific antivenoms . Brit. med. J. 4, 137. WARRELL, D. A. aIld ARNErT, C. (1976) The importance of bites by the saw-scaled or carpet viper (Echis carinatus) : epidemiological studies in Nigeria and a review of the world literature . Acto trop. 33, 307.
0041-0101/79/0901-031IS02.00/0
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) IN ASSESSING ANTIVENOM POTENCY R. D. G. TIiEAKSi'ON and H. A. Re1n
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, United Kingdom (Accepted for publication 5 March 1979) ABSTRACT R. D. G. THFAK31'O1V and H. A. Rte. Enzyme-linked immunosorbent assay (ELISA) in assessing antivenom potency. Toxicon 17, 511-515, 1979.-Enzyme-linked immunosorbent assay (ELLSA) has been investigated as a screen of antivenom potency. One batch from each of three commercial sotuces and from two rabbit research antivenoms were established as the five group reference antivenoms and the median effective doses (En bos) for neutralising four venoms medically important in Africa were estimated in mice. Reference curves were prepared relating EDbo s to optical densities with ELISA . The En 6os of 38 different batches of test antivenom were estimated by ELISA, and ELISA eo, os were then compared with in vivo mouse ensos. Excellent correlation was obtained . The method is rapid, cheap, simple, economic in amounts of venom used, and most importantly, greatly reduces the need for live animals. If validated in an extended range of venoms and antivenoms, ELISA screening of antivenom potency should be a useful standard procedure. TNTRODUCTION
assays of biological substances such as antivenoms, it is highly desirable to avoid suffering and death ofanimals by substituting in vitro methods, provided such methods measure life-saving capability with precision similar to that of in vivo methods. liut in vitro methods so far tried have not been successful (CHRISTENSEN, 1968) ; we have therefore investigated enzyme-linked immunosorbent assay (ELISA) in this problem (THEAKSTON et al., 1977). Irr PorErrcY
MATERIALS AND METHODS Venoms and antivenoms Pooled representative samples of four venoms medically important in Africa were obtained by personally milking adult specimens of the puff adder (Batas arietans), carpet viper (Echis carinatus), Egyptian cobra (Naja haje) and spitting cobra (Naja aigricollis). All the snakes were maintained in our herpetarium and were kindly supplied from north Nigeria by Drs. R. N. H. Pugh and D. A. Walrell. Venom was weighed, freeze-dried and stored in the dark at 4°C. Monospecific antisera were raised in rabbits against B. arietans and E. carinatus venoms (Txsnxsrorr et al ., 1977). Relevant commercial therapeutic antivenoms (Tables 1 and 2) were from Behringwerke AG (Behr), Postachliessfach 167, West Germany (kindly donated) ; the South African Institute for Medical Research (SAIMR), P.O . Boz 1038, Johannesburg ; and the Haffkine Institute (HTj, Parel, Bombay l2, India. Behr and SAIMR antivenoms are fluid; HI antivenom is freezedried. Outdated antivenoms from these throe institutes were kindly donated by the Zoological Society of London. All batches of antivenom were kept in the dark at 4°C until use. The investigations reported in this paper were performed during April-November 1978 . Lethal toxicity of venoms
Lethal toxicity was assessed by subcutaneous (s .c .) injection of venom in 02 ml physiological saline into the hind thigh of mice weighing 18-20 g. Six mice were used at each venom dose . The median lethal dose (tn, o) was calculated by probat analysis (Fnvtv>:v, 1971) of deaths occurring within 24 hr of venom injection. Lobos and 95 ~fiducial limits in parentheses were 1701tg/mouse (140-210) for B. arietansvenom,120 ug/mouse (84-150) for E. carirtatus venom, 14 ug/mouse (13-16) for N. haje venom and 106 ug/mouse (80-120) for N. rrigrtcollis venom.
51 2
R. D. G. THEAICSTON and H. A. REID
Estlrnation ojaruivenam neutralising ED bo F1Ve LD6oS of venom in 0~2 ml of physiological saline was injected s.c . into mice and then varying amounts of antivenoms in 7 graded doses (200~~25 itl) made up to a total of 0~2 ml in physiological saline, were injected rapidly into the tail vein of mice (6 mice per dose). After observing the mice for 24 hr, the neutralising median effective dose (®bu) and 95 ~ fiducial limits were calculated by probit analysis. The ®bn is the least amount of antivenom needed to prevent death of half the mice in a group (thus, the lower EDbu, the higher the neutralising ability) . If 200 ul antivenom failed to save at least half the mice, the antivenom was judged ineffective. Group reference antivenoms One batch of antivenom from each of the three institutes and from a pooled batch of the two monospecific rabbit antivenoms were arbitrarily selected as Group Reference and ED bos against each of the four venoms were determined . A range of multiples and fractions of these EDbu s, from 6 times the EDbp down to 1/64th EDba, were diluted 1/50 (commercial antivenoms) and I/1000 (rabbit antivenoms) in PBS "Tween" and assayed for each of the four relevant antibodies using ELISA (Ti1EAICSTON et al., 1977). The crude rabbit antivenoms were more diluted than the refined commercial antivenoms because the former give a much higher optical density with ELISA than the latter at a given ED bo . Commercial antivenoms were incubated with substrate for 40 min, rabbit antivenoms for 20 min. Table 1 shows the ED6o of each reference antivenom and its corresponding optical density with ELLSA. All assays were in duplicate. Group reference curves of optical densities from ELISA were then plotted against the mouse in vtvo ED6 u for each of the four venoms . TABLE l . NEUTRALISING ED6 pS AND ELISA READINGS Antivenom Source, batch No. Behringwerke 317 SAIMR 0938 Haffkine 135-E Rabbit R6 Rabbit R5
Labelled active against" BAV ECV NHV NNgV BAV ECV NHV NNgV ECV (N. naja)
Expiry date (month/yr) 8/77 7/75 10/74
Venom tested BAV ECV NHV NNgV BAV ECV NHV NNgV ECV NHV NNgV BAV ECV
OF
REPERENCE ANTIVENOMS eDba (lil/mouse)t 64 ( 29-121) 34 ( 15- 66) 192 (100-350) 182 ( 95-340) 22 ( 8- 46) 16 ( 6- 36) 35 ( 16- 68) 48 ( 22- 89) 50 ( 22- 92) 144 ( 75-230) 161 ( 85-299) 35 ( 16- 72) 64 ( 29-121)
ELISA optical density at 400 nm 0700 0520 0295 0l42 0206 0335 0l36 0089 0530 0256 0l37 098l 1012
'Venoms : BAV = Bais arletans ; ECV = Echis carinates ; NHV = Naja haje ; NNgV = Naja nigricolhs . tNumbers in parentheses are 95 ~ fiducial limits . Investigation of unknown antivenoms By comparing (spectrophotometrically) the optical densities of different doses of unknown antivenoms (200-3125 p1 diluted 1/50 in PBS "Tween") with those of the relevant reference curve at 1 ED 6o, the theoretical ELISA ED 6, was obtained . The same conditions applied with crude rabbit antivenoms except that 1/1000 dilutions were used . In vivo ED bos were then determined in mice, using 7 graded doses ranging from 200 to 625 IIl with 6 mice at each dose . The results were compared with ELISA EDb,s and optical density/ED6 u curves were collated with relevant reference curves . RESULTS
The close correlation between the ELISA ED bo and mouse in vivo ED 6o is shown in Fig . 1 and in Table 2. For 16 E. carinatus venom rabbit antivenoms the correlation coefficient was 097 (P < 0001) ; similar results were obtained with 10 BAV rabbit antivenoms. With the 12 test batches of commercial antivenoms against E. carinatus venom, correlation was again highly significant (r = 092 ; P < 0001). ELISA optical density/mouse ED6o curves of test antivenoms and their appropriate reference curves were very similar ; Behr and HI curves were almost identical whereas SAIMR gave lower optical densities and crude rabbit antivenom showed a much more intense colour reaction.
51 3
ELLSA and Antivenom Potency
R ioo
0 W
In vivo eo yo , wl
Z(~Î
FiO. 1. CORRELATION BETWEEN NEIJTRALL41N0 ED S p FROM ELISA AND MOUSE In VIVO ED~p (r = 0'96 ; P < U~001) .
DISCUSSION
Most ofthe commercial antivenoms investigated were `outdated' atthe time we tested them and the neutralising potency found in our tests confirms the conclusion by CHRISTENSBN (1975) that it is unrealistic and wasteful to limit the `life' of refrigerated refined antisera to 5 yr. Christensen also noted that opacities developed before potency was lost. All the antivenoms we tested were clear, except SAIMR batch 1323 (which nevertheless retained significant neutralising ability despite having opacities) . In general, SAIMR antivenoms showed the most potent neutralising ability and this has been confirmed clinically for E. carinatus venom in man (WARRELG Ct aL, 1974). The ability of SAIMR antivenom 1879, labelled as active only against E. carinatus venom to neutralise other venoms can be explained by previous immunisation ofthe horses concerned with other venoms (CHRISTENSEN, personal communication, 1978). The poor neutralising ability of Echis antivenom against E. carinatus venom from different geographic areas has also previously been observed both clinically (WARRELL and ARNETT, 1976) and in the laboratory (LATIFI, 1973). ELISA measures antibody (or antigen) and we realise that antigen-antibody reactions of venoms and their degree of toxicity are two distinct and sometimes unrelated things (SCHÖTTLER, 1951). Antigens may be nontoxic and toxins may be non-antigenic. Nevertheless, our preliminary studies suggest sufficient correlation between the in vivo mouse EDao and the ELISA optical density to warrant further exploration of ELISA as a simple in vitro screening assay, both ofcommercial therapeutic antivenoms and of experimental antisera . At present the ELISA reference standard has to be separately established for each commercial group or each source . This may be due to different methods of production or purification ; certainly crude antivenoms give a much higher optical density with ELISA at a given En so . We can screen 5 antivenoms from one commercial group on one ELISA plate by simply placing on the plate the known group reference at 1 x aD6o and different dilutions of the 5 test antivenoms . The point of colour match (optical density) between the reference antivenoms and the test dilution, enables the quick calculation ofthe Ens o of the test antivenom. Compared with in vivo potency tests, ELISA is simpler (if a spectrophotometer is not
51 4
R. D. G. THEAKSTON and H. A. REID TABLE
2.
NEUTRALISINQ ED6pS
Antivenom Labelled active Source, batch no. against" Behringwerke BAV 1472 NHV NNgV Behringwerke 1497 Behringwerke 1527 Behringwerke 1575 Behringwerke 1576
BAV ECV NHV NNgV BAV ECV NHV NNgV ECV NHV
(ELISA
Expiry date (month/yr) 2/72 1/73 12/73 8/75
10/81
SAIMR 1323
BAV ECV NHV NNgV BAV ECV NHV NNgV BAV Naja sp.
SAIMR 1773
BAV Naja sp .
12/75
SAIMR 1879
ECV
5/80
SAIMR 2325
BAV Naja sp .
6/80
Haffkine 420-D
ECV (N. naja)
2/81
Haffkine 446-G
ECV (N. naja)
8/81
Behringwerke 207018
AND In VIVO MOUSE) OF TEST ANTIVENOMS
9/75
10/72
Venom tested" BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV BAV ECV
NHV NNgV BAV ECV NHV NNgV BAV ECV NHV NNgV ECV NHV NNgV ECV NHV NNgV
ELLSA Eubo
(1tl/mouse) 46 47 175 122 nil 75 110 60 57 23 117 nil 97 46 l25 nil 67 48 107 nil 23 22 200 100 47 90 25 142 23 200 40 50 50 9 21 110 21 30 30 67 100 nil nil 100 nil nil
//1 l'il'O EDSp
(ul/mouse)fi 40 ( l9- 73) 60 ( 35- 98) 200 (140-350) l19 ( 52-230) nil 70 ( 32--138) 122 ( 46-356) 100 ( 54-180) 63 ( 27-109) 28 ( 13-- 56) 160 ( 88-350) nil 90 ( 38-145) 49 ( 22- 89) 13G ( 72-270) nil 68 ( 31-115) 55 ( 25- 96) 200 (120-360) nil 22 ( 10- 45) 32 (15- 64) nil 137 (104-199) SO ( 24- 99) 74 ( 36-120) 28 ( 13- 56) 150 ( 78-260) 28 ( 12- 57) 160 ( 84-290) 35 ( 15- 66) 49 ( 23- 87) 25 ( l2- 54) l3 ( 4-- 32) 75 ( 39-140) 150 ( 80-290) 20 ( 9- 42) 40 (19- 73) 31 ( 14- 59) 69 ( 31-113) 87 ( 39-165) nil nil 83 ( 36-134) nil nil
"Venoms : BAV = Bltis arietans ; ECV = Echls carlnatus; NHV = Naja haje ; NNgV = Naja nigricollis . tNumbers in parentheses are 95 ~ fiducial limits .
available, antivenoms can be assayed with experience to the equivalent of 0050 optical density by visual matching) and more rapid . The economy in venom and, more importantly, in animals is notable . We are currently extending ELISA screening of antivenom potency to other venoms and antivenoms with a view to validating the technique as a standard procedure . Acknowledgements-We thank Miss S. B. J.
MACFARLANE
for advice on statistics, Mrs. D.
InnoN
and Miss A.
ELLSA and Antivenom Potency
S15
RItNMER for technical help, Mrs. HEATHER HUXLEY for secretarial work, and the Ministry of Overseas Development and the Medical Research Council for financial support. REFERENCES CHRLSTENSEN, P. A. (1968) The venoms of central and south African snakes . In : Venomous Animals and Their Venoms, Vol . 1, Ch . 16, p. 437, (BUCHERL, W., BUCKLEY, E. and DELOFEU, V. Eds.) . New York : Academic Press. C.HRI3TENSEN, P. A. (1975) The stability of refined antivenin. Toxlcon 13, 75 . FINNEY, D. J. (1971) Probit Analysis, 3rd edition. Cambridge. LArtrt, M. (1973) Studies of the venom of Iranian Echis carinatus in comparison with those of Pakistan and Eritria . Proc. 9th lnt. Cong. Tropical Medicine and Malaria, Athens, 14-21 October. Abstract 107. SCHÖITLER, W. H . A. (1951) Antigen-antibody relations in the present antivenin production of Brazil .
Am . J. trop . Med. 31, 500 .
THEAKSiON, R. D. G., LLOYD-JONES, M. J. and REID, H. A. (1977) Micro-ELISA for detecting and assaying snake venom and venom-antibody. Lancet ü, 639. WARRELL, D. A., DAVIDSON, N.MCD ., ORMEROD, L. D., POPE, H. M., WATKINS, H. M., GREENWOOD, B. M. and REID, H. A. (1974) Bites by the saw-scaled or carpet viper (Echis carinatus) : trial of two specific antivenoms . Brit. med. J. 4, 137. WARRELL, D. A. aIld ARNErT, C. (1976) The importance of bites by the saw-scaled or carpet viper (Echis carinatus) : epidemiological studies in Nigeria and a review of the world literature . Acto trop. 33, 307.