Journal of immunological Methods. 146(1992) 213-218
213
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0022-1759/92/$05.00
JIM06177
A novel format for a rapid sandwich EIA and its application to t h e identification of s n a k e v e n o m s J o h n C. Cox, A n a s t a s i a V. Moisidis, J a n e M. S h e p h e r d , D e b b i e P. D r a n e a n d S t e p h e n L. J o n e s lmmunodiagnostics Section, Research and Decelopment Dit'ision, Commonwealth Serum Laboratories Limited, Parkville. Victoria 3052, Australia
(Received20 May 1991, revisedversionreceived20 September 1991,accepted 30 September 1991)
A rapid sandwich enzyme immunoassay format is described where conjugate is lyophilised within the well in which the test reaction will occur. The format is straight forward to manufacture, has a prolonged shelf life, and eliminates one incubation and wash step from the usual test procedure. The technology has been applied to the development of a rapid assay for the identification of snake venom in clinical specimens. The resultant assay was specific and sensitive, provided rapid results and was appropriate for field use. Key words: Novel field assay; Enzyme immunoassay;Snake venom identification
Introduction
There is an increasing requirement for rapid diagnostic assays which can be used by clinicians in their offices and veterinarians and agriculturalists in the field. It is important that such tests be accurate and inexpensive, occupy minimal storage space and be rapid and simple to perform. In situations where speed and high sensitivity are required, enzyme immunoassay (EIA) technology has generally beel~ chosen. The solid phase for these assays has usually been either a plastic (generally polystyrene) microtitre well or a nitrocellulose (or nylon) membrane (Tijssen, 1985), although other surfaces such as glass capillaries have also been described (Chandler and Hurrell, 1982). Each system has advantages and draw-
Correspondence to: J.C. Cox, CommonwealthSerum Laboratories Limited, 45 Poplar Road, Parkville, Victoria 3052, Australia.
backs. Microwell tests are the least expensive to manufacture but are the most complex tests to perform. Membrane based tests, although requiring the same number of steps, are simpler mainly because each wash is reduced to a single step. They are, however, expensive to produce and not particularly suited to quantitation or to multiple determinations on a single specimen. The glass capillary format is easy to use but very expensive to manufacture. The venoms from Australian snakes fall into five broad groups based upon the most appropriate antivenom for treatment of the envenomed patient. The ability to assign a venom to one of these five groups will permit use of a monovalcnt antivenom with a concomitant cost saving and reduced total dosage of foreign immunoglobulin. In the absence of this knowledge, in most states of Australia a polyvalent antivenom must be used (Sutherland, 1983, 1990). The first radioimmunoassay to detect snake venom was developed by Coulter et al. (1974) and
214 its clinical and forensic value was soon established (Sutherland et al., 1975). Coulter et al. (1978) further showed that reliable identification of snake venoms could be achieved in a sandwich radioimmunoassay which used antisera of the same specificity for both the capture and conjugate antibody. The analogous EIA was made available for clinical use (Coulter et al., 1980), and although it established a need for such a test, its performance was too complex to gain widespread acceptance in the field. This was because the test kit comprised five separate conjugates, one for each of the five major antivenom groups and the kit user Was required to add a dilution of conjugate 1 to well 1, a dilution of conjugate 2 to well 2 and so on. A glass capillary format was devised (Chandler and Hurrell, 1982) to make the EIA suitable for routine field application and although difficult to produce, had good field acceptability (Hurrell and Chandler, 1982). However, the glass capillary format required the use of a single mixture of conjugates, and because of this it was necessary to affinity absorb the solid-phase antibodies to generate the required specificity. Subsequent extensive use in the field has highlighted several anomalies where the venom from less common snakes can be assigned incorrectly to one of the five groups. Most importantly, venoms from Red-bellied Black (Pseudechis porphyriacus) and Copperhead (Austrelaps superba) snakes reacted equally in both Tiger and Black categories but are most appropriately identified as Black and Tiger respectively. Additionally, venom of the Clarence River snake (Tropidechis carinatus) reacted with both Tiger and Taipan whereas it correctly belonged in the former group. These anomalies were unique to the new glass capillary format test (Coulter, unpublished results). The new format (COx, 1988) which was developed to resolve the above problems, permits the use of a different conjugate for each well by lyophilising conjugate within the appropriate well at the time of manufacture. As a result affinity absorption of the capture antibody is unnecessary and, because a simultaneous assay results, the steps normally required for conjugate incubation and washing are eliminated from the assay procedure.
Materials and methods
Antisera to snake venoms Antibodies to the venoms of the five snakes of greatest clinical importance in Australia, Brown ( Pseudonaja textilis), Death Adder ( Acanthophis antarcticus), King Brown (Pseudechis australis), Taipan (Oxyuranus scutellatus) and Tiger (Notechis scutatus), were produced in rabbits. These venoms and certain others were obtained from the Australian Reptile Park, Gosford, Australia and Mr. C. Tanner, Cooktown, Australia. Rabbits were injected with graded doses of venom from 10 /~g to 2 mg at 2-weekly intervals. The first four doses were in Freund's complete adjuvant (Commonwealth Serum Laboratories Limited (CSL), Parkville, Australia) and delivered intramuscularly deep into the hind or foreleg. Subsequent doses were delivered subcutaneously in Freund's incomplete adjuvant (CSL). Sera from test bleeds taken during the immunization course and large bleeds taken 1 week after the final dose were tested by gel diffusion. Selected rabbits were boosted and bled at six week intervals. IgG was purified from rabbit antisera by protein A chromatography according to the method of Goding (1976).
Preparation of conjugates Preparations of IgG were labelled with horseradish peroxidase according to the procedure of Wilson and Nakane (1978). At completion of the reaction conjugates were diluted to 1 mg/ml in a suitable conjugate stabilising buffer and held at 4°C.
Preparation of plates Polystyrene microtitre plates with 8 well removable strips (MaxiSorp, Nunc, Denmark) were used. Coating antibodies were diluted to 2/zg/ml in 0.05 M carbonate buffer, pH 9.6, and 100/~i added to each well of the 96 well plate. Following incubation overnight at 4°C, unbound antibodies were removed by aspiration and unreacted sites blocked by incubation with 150 /~! of 1 mg/mi casein in PBS Tween (0.01 M phosphate-buffered saline, pH 7.2 (PBS), containing 0.05% (v/v) Tween 20) at 20°C for 1 h. The blocking solution was then aspirated and replaced with a 5% (w/v)
215 solution of lactose in water for 5 min at 20°C in order to stabilise bound antibodies. After aspiration, 10/~1 of conjugate in stabilising buffer containing 0.15% (w/v) alphazurine A (an inert blue dye) were added. Plates were immediately transferred to the shelves of a freeze drier and lyophilised with secondary drying over P205 . Individual 8-well strips were capped, sealed in laminated foil pouches containing a sachet of silica gel and held at 4°C unless required for stability trials.
Experimental assay procedures Sequential and simultaneous assay procedures were compared using plates prepared as described above but vacuum dried at room temperature without the addition of conjugate. Venom samples were diluted in Yellow Diluent, a PBS Tween formulation containing 10 m g / m l casein and 40 /xg/ml tartrazine (an inert yellow dye). For the sequential assay, 50 /~1 Yellow Diluent were added to each well followed by 50/.d of test sample. The contents were mixed and incubated at room temperature for the specified time. The wells were then washed seven times with PBS Tween and tapped dry. Diluted conjugate (100 /~1) was added to each well and incubated and washed as before. Finally, 50 p.I each of peroxide solution (2 × ) and chromogen solution (2 × ) (tetramethylbenzidine; Bos et al., 1981) were added to each well. The contents of the wells were gently mixed and after 10 min the reaction was terminated by addition of 50 /~1 0.5 M H2SO 4. Absorbance values were read at 450 nm. For the simultaneous assay, 50 p.! of test sample and 50 p.l of conjugate at twice the concentration used above were added to each well, gently mixed and incubated at room temperature for the specified time. The wells were then washed and peroxide and chromogen solutions added as described above.
Kit components and performance The kits comprised an eight-well microtitre strip containing lyophilised conjugate and blue dye, Yellow Diluent, chromogen solution (2 x concentrate) and peroxide solution (2 x concentrate). Addition of the test sample in sample diluent to the microtitre wells reconstituted the
conjugate which could be visualised by the operater as the generation of a uniform green colour on mixing. Test sample was added to seven wells of an eight-well strip. The first well contained capture antibody and conjugate to Tiger snake venom (TSV) and similarly the next four wells contained reagents to identify venoms from Brown snake, Black snake, Death Adder and Taipan. The sixth well was a negative control with normal rabbit lgG as capture and an antioTSV conjugate. The seventh well was a positive control with normal rabbit lgG as capture and a sheep antirabbit lgG conjugate. The final well was blank (neither capture antibody nor conjugate) and could be used to orientate the strip. Following a 10 min incubation with sample at room temperature, the wells were washed at least seven times under running tap water. Then one drop each of chromogen solution and peroxide solution were added to each well and gently mixed. Wells were observed for the development of a blue colour, which occurred within 10 min for positive test samples and the positive control. The negative control well should remain colourless.
Stability trials Individually sealed strips were held at -70°C, 4°C, 18°C and 37°C. At intervals, pouches were allowed to equilibrate to room temperature, opened and tested. Results were calculated as the percentage drop in absorbance, assuming a 100% retention of activity at -70°C.
Results
Preliminary experiments were performed to compare the sensitivity of venom detection in sequential and simultaneous assay format. An incubation time of 10 min was used for the simultaneous assay and both 2 x 5 rain and 2 x 10 min incubations for the sequential assay. The results at various levels of TSV from 1.25 to 80 n g / m l are shown in Table I. It can be seen that for a given assay time, i.e., 1 x 10 min simultaneous or 2 x 5 rain sequential, the simultaneous assay was more sensitive at all points of the clinically rele-
216 TABLE I COMPARISON OF SIMULTANEOUS AND SEQUENTIAL SANDWICH EIA FORMATS ON SNAKE VENOM ASSAY SENSITIVITY Assay format
Concentration of Tiger Snake Venom (TSV) (ng/ml) 0 1.25 2.5 5 10
20
40
80
Simultaneous (1 × 10 min) Sequential (2×5 rain) Sequential (2 x I0 rain)
0.067 a 0.061 0.062
1.145 0 ~t,:5 0.7t"9
1.684 0.627 1.329
2.360 1.033 2.078
0.152 0.077 0.098
0.236 0.098 0.141
0.392 0.135 0.286
0.748 0.202 0.493
a Absorbance reading at 450 nm after addition of 0.5 M H2SO4.
v a n t r a n g e o f v e n o m c o n c e n t r a t i o n s a n d easily able to d e t e c t 2.5 n g / m l v e n o m . E x p e r i m e n t s were n e x t p e r f o r m e d to d e t e r m i n e t h e o p t i m a l v o l u m e o f c o n j u g a t e to lyophilise in e a c h well a n d to e s t i m a t e t h e loss o f activity d u r i n g lyophilisation. V o l u m e s o f 5, 10 a n d 20/~1 o f c o n j u g a t e p e r well were investigated, e a c h at c o n c e n t r a t i o n s covering t h e r a n g e f r o m 0.2 to 2.0 / t g c o n j u g a t e p e r well. T h e pellet r e s u l t i n g f r o m lyophilisation o f 20 /.d w a s f o u n d to b e insufficiently stable to i m p a c t w h e r e a s t h a t r e s u l t i n g f r o m 5 a n d 1 0 / t l w a s u n a f f e c t e d by jolting well in excess o f t h a t n o r m a l l y e x p e c t e d to occur. A 10 /~! d i s p e n s e d v o l u m e at 0 . 2 - 0 . 4 / ~ g c o n j u g a t e p e r
well (i.e., 2 0 - 4 0 p g / m l ) was finally c h o s e n as t h e o p t i m u m condition. L o s s e s o f a r o u n d 2 0 % o f c o n j u g a t e activity w e r e o b s e r v e d d u r i n g lyophilisation. T o d e t e r m i n e t h e sensitivity a n d specificity o f t h e assays, v e n o m s f r o m t h e five c o m m o n s n a k e s w e r e d i l u t e d to 10 a n d 40 n g / m l in d i l u e n t b u f f e r a n d t e s t e d as p e r instructions. T h e r e s u l t s o f o n e typical e x p e r i m e n t (Table II) s h o w e d t h a t all five v e n o m s w e r e unequivocally identified at 10 n g / m l (a distinct b l u e c o l o u r c o r r e s p o n d i n g to a n abs o r b a n c e r e a d i n g g r e a t e r t h a n 0.2) a n d t h a t cross-reactions, t h o u g h p r e s e n t , were c o n s i d e r ably w e a k e r t h a n specific reactions. Additionally,
TABLE II REACTION OF 5 COMMON AND 7 LESS COMMON VENOMS IN THE SNAKE VENOM DETECTION TEST Test venom
Tiger Brown King Brown Death Adder Taipan Clarence River Copperhead Palehead Dugite Gwardar Papuan Black Red-bellied Black
Concentration ng/ml
Tiger
Specificity of test well Brown
Black
Death Adder
Taipan
Neg.
Dos.
10 40 10 40 10 40 10 40 10 40 40 40 40 40 40 40 40
0.65 a 1.95 0.10 0.13 0.24 0.57 0.16 0.25 0.19 0.40 1.08 0.44 0.28 0.12 0.12 0.20 0.21
0.20 0.20 0.38 0.91 0.17 0.27 0.15 0.19 0.18 0.24 0.28 0.18 0.15 0.48 0.45 0.14 0.13
0.13 0.18 0.09 0.14 0.47 1.38 0.11 0.13 0.13 0.12 0.15 0.28 0.20 0.10 0.10 0.61 0.34
0.12 0.20 0.08 0.11 0.15 0.34 0.40 0.92 0.11 0.16 0.12 0.25 0.16 0.10 0.09 0.21 0.22
0.14 0.24 0.12 0.20 0.15 0.36 0.12 0.23 1.12 2.32 0.23 0.23 0.17 0.13 0.13 0.17 0.17
0.10 0.09 0.09 0.15 0.09 0.20 0.09 0.12 0.11 0.13 0.09 0.08 0.09 0.11 0.11 0.08 0.08
2.06 2.21 2.05 2.16 2,02 2.39 1.91 2.30 1.57 2.09 1.87 2.06 !.96 1.92 1.94 1.99 2.16
a Absorbance reading at 450 nm after addition of 0.5 M H2SO4.
217 seven less common venoms (listed in Table II) were tested at 40 ng/ml (a concentration which approximates to that of a bite-site sample) and the venom to which they bore the closest antigenic resemblance was determined. In six of these seven cascs, this indicated the most appropriate antivenom for treatment of bites. In the other case, Red-bellied Black was grouped correctly as a Black snake (it is genus Pseudechis as are other common members of this group), even though its bite is more appropriately treated with TSV antivenom (Sutherland, 1990). The stability of strips stored at 4°C, 18°C and 37°C was estimated at intervals from 1 to 40 weeks and compared with results obtained for strips stored at - 7 0 ° C. Wells were reacted with a low level (2.5-5 ng/ml) and a high level (40 ng/ml) of TSV and the results calculated as the percentage absorbance at the stated temperature relative to the - 70 ° C wells. Strips stored at 4°C lost around 15% of their activity after 40 weeks relative to strips stored at -70°C and were still able to detect reliably 2.5 ng/ml venom. Accelerated stability trials showed that strips retained 75% of their activity after 40 weeks at 18°C and 31% of their activity after 40 weeks at 37°C.
Discussion An essential requirement for the novel assay described in this paper is that the antibodies selected for the kit development must be able to function in a simultaneous assay format. The complex nature of the various antigens involved in the snake venom detection assay and the need to use polyclonal rather than monoclonal antibodies made the expected outcome uncertain. The results (Table I) show that a single incubation step of 10 rain in a simultaneous assay results in a more sensitive assay than two incubation steps, each of 5 or 10 min in a sequential assay. Thus, the simultaneous assay was both quicker and more sensitive. Similar benefits have been observed by us when developing assays for canine parvovirus in dog faeces (Drane et al., unpublished observations), human interferon-~, and hu-
man blood grouping (Isabel Roberts, personal communication). The observation that the simultaneous assay was both quicker and more sensitive can possibly be explained by a build-up of antigen-conjugate complexes bound to the solid phase in a simultaneous assay. Such multi-layer complexes cannot form in a sequential assay. The other consideration of note in developing this new assay concept was to iyophilise the conjugate within the well so that it did not interfere with the stability of the capture antibody, retained the stability of lyophilised enzyme conjugates and could not readily be dislodged from the well by physical shock. None of these issues in reality caused a problem. Typical losses o[ 20% of conjugate activity during drying were compensated for by an appropriate adjustment in conjugate strength. Volumes of 5 and 10/zi resulted in pellets which adhered satisfactorily to the surface of the wells, and the addition and lyophilisation of conjugate did not interfere with the activity or stability of the capture antibody. This was reflected in the excellent stability observed for the kit. To permit calculation of stability of the snake venom detection kit, it was decided to use as the reference wells stored at -70°C after lyophilisation. In this way, day to day variations due to temperature, sample preparation and other assay variables including stability of other reagents were removed from the calculation. The possibility of an initial sharp drop of activity in reference wells as a result of the transition from 4°C to -70°C was excluded by initial stability data which showed equal sensitivity for the -70°C and 4°C wells. The results suggest a useful reagent life in excess of 2 years. Extensive field evaluation of the snake venom detection kit for both veterinary and human application is in progress. Preliminary evaluations again show the new kit format to be simpler to use and more sensitive and specific. This increased specificity is particularly demonstrated by the ability of the test to identify correctly the most appropriate antivenom for treatment of the envenomed patient following bites by a number of less common snakes (Table II). Bites by these snakes have led to some confusion in interpretation of test results using the glass capillary assay kit.
218 T h e n e w t e c h n o l o g y d e s c r i b e d h e r e c a n have definite a d v a n t a g e s e v e n in a simple diagnostic test s i t u a t i o n s u c h as d e t e c t i o n o f specific virus in a faecal s a m p l e . However, in m o r e c o m p l e x test situations, especially w h e r e a biological s p e c i m e n is to be t e s t e d for t h e p r e s e n c e o f a n u m b e r o f possible analytes, for e x a m p l e t h e identification o f s n a k e v e n o m , t h e test c o n c e p t offers m a j o r a d v a n t a g e s in a d d i t i o n to e a s e a n d s p e e d o f perf o r m a n c e . M o s t importantly, a n t i s e r a for t h e diff e r e n t a n a l y t e s c a n b e derived f r o m d i f f e r e n t species b e c a u s e t h e n o r m a l p r o b l e m s a s s o c i a t e d with a m i x e d s p e c i e s c o n j u g a t e a r e avoided. F u r t h e r m o r e , a n y blocking p r o c e d u r e w h i c h m a y b e n e c e s s a r y for a n y specific r e a c t i o n c a n be conf i n e d to t h a t well by inclusion in t h e c o n j u g a t e stabilising b u f f e r for t h a t c o n j u g a t e . T h e relative sensitivity a n d specificity o f e a c h well c a n also b e a d j u s t e d b o t h by variation in t h e a n t i b o d y concentration of the capture and the conjugate phase. Finally, only r e l e v a n t c o n j u g a t e is p r e s e n t in e a c h well. A s a result, c o n j u g a t e cost is less a n d , m o r e importantly, b a c k g r o u n d reactivity w h i c h is largely i n f l u e n c e d by t h e a m o u n t o f c o n j u g a t e p r e s e n t , is significantly r e d u c e d . T h e a d v a n t a g e s o f t h e n e w r a p i d assay f o r m a t d e s c r i b e d h e r e s u g g e s t f u r t h e r laboratory a n d field applications. O f m a j o r interest a r e solid p h a s e blood g r o u p serology, m e a t s p e c i e s identification a n d r a p i d t e s t s for identification o f respiratory a n d intestinal p a t h o g e n s . D e v e l o p m e n t o f t h e s e t e s t s is u n d e r w a y .
References Bos, E.S., Van der Doelen, A.A., Van Rooy, N. and Schuurs, A.H.W.M. (1981) 3,Y,5,5'-Tetramethylbenzidine as an
Ames test negative chromogen for horseradish peroxidase in enzyme-immunoassay. J. Immunnassay2, 187. Chandler, H.M. and Hurrell, J.G.R. (1982) A new enzyme immunoassaysystem suitable for field use and its application in a snake venom detection kit. Clin. Chim. Acta 121, 225. Coulter, A.R., Sutherland, S.K. and Broad, A.J. (1974) Assay of snake venoms in tissue fluids. J. ImmunoL Methods 4, 297. Coulter, A.R.. Cox, J.C., Sutherland, S.K. and Waddell, CJ. (1978) A new solid-phase sandwich radioimmunoassay and its application to the detection of snake venom. J. ImmunoL Methods 23, 241. Coulter, A.R., Harris, R.D. and Sutherland, S.K. (1980) En~ m e immunoassay for the rapid clinical identification of snake venom. Med. J. Aust. i, 433. Cox, J.C. (1988) Improved immunoassay. International Patent Application no. PCT/AU89/00503. Goding, .I.W. (1976) Conjugation of antibodies with fluorochromes: Modifications to the standard methods. J. Immunol. Methods 13, 215. Hurrell, J.G.R. and Chandler, H.M. (1982) Capillary enzyme immunoassayfield kits for the detection of snake venom in clinical specimens. A review of two years use. MOd. J. Aust. 2, 236. Sutherland, S.K. (1983) Australian Animal Toxins. The Creatures, Their Toxins and Care of the Poisoned Patient. Oxford University Press, Melbourne. Sutherland, S.K. (1990) Treatment of snake bite. Aust. Fam. Physician 19, 21. Sntherland, S.K., Coulter, A.R., Broad, AJ., Hilton, J.M.N. and Lane, L.H.D. (1975) Human snake bite victims: The successful detection of circulating snake venom by radioimmunoassay. Med. J. Aust. 1, 27. Tijssen, P. (1985) In: R.H. Burden and P.H. Van Knippenberg (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, Vol 15, Practice and Theor~ of Enzyme Immunoassays. Elsevier, Amsterdam, p. 297. Wilson, M.B. and Nakane, P.K. (1978) Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In: W. Knapp, K. Holubar and G. Wick (Eds.), Immunofluorescence and Related Staining Techniques. Elsevier, Amsterdam, p. 215.
213
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0022-1759/92/$05.00
JIM06177
A novel format for a rapid sandwich EIA and its application to t h e identification of s n a k e v e n o m s J o h n C. Cox, A n a s t a s i a V. Moisidis, J a n e M. S h e p h e r d , D e b b i e P. D r a n e a n d S t e p h e n L. J o n e s lmmunodiagnostics Section, Research and Decelopment Dit'ision, Commonwealth Serum Laboratories Limited, Parkville. Victoria 3052, Australia
(Received20 May 1991, revisedversionreceived20 September 1991,accepted 30 September 1991)
A rapid sandwich enzyme immunoassay format is described where conjugate is lyophilised within the well in which the test reaction will occur. The format is straight forward to manufacture, has a prolonged shelf life, and eliminates one incubation and wash step from the usual test procedure. The technology has been applied to the development of a rapid assay for the identification of snake venom in clinical specimens. The resultant assay was specific and sensitive, provided rapid results and was appropriate for field use. Key words: Novel field assay; Enzyme immunoassay;Snake venom identification
Introduction
There is an increasing requirement for rapid diagnostic assays which can be used by clinicians in their offices and veterinarians and agriculturalists in the field. It is important that such tests be accurate and inexpensive, occupy minimal storage space and be rapid and simple to perform. In situations where speed and high sensitivity are required, enzyme immunoassay (EIA) technology has generally beel~ chosen. The solid phase for these assays has usually been either a plastic (generally polystyrene) microtitre well or a nitrocellulose (or nylon) membrane (Tijssen, 1985), although other surfaces such as glass capillaries have also been described (Chandler and Hurrell, 1982). Each system has advantages and draw-
Correspondence to: J.C. Cox, CommonwealthSerum Laboratories Limited, 45 Poplar Road, Parkville, Victoria 3052, Australia.
backs. Microwell tests are the least expensive to manufacture but are the most complex tests to perform. Membrane based tests, although requiring the same number of steps, are simpler mainly because each wash is reduced to a single step. They are, however, expensive to produce and not particularly suited to quantitation or to multiple determinations on a single specimen. The glass capillary format is easy to use but very expensive to manufacture. The venoms from Australian snakes fall into five broad groups based upon the most appropriate antivenom for treatment of the envenomed patient. The ability to assign a venom to one of these five groups will permit use of a monovalcnt antivenom with a concomitant cost saving and reduced total dosage of foreign immunoglobulin. In the absence of this knowledge, in most states of Australia a polyvalent antivenom must be used (Sutherland, 1983, 1990). The first radioimmunoassay to detect snake venom was developed by Coulter et al. (1974) and
214 its clinical and forensic value was soon established (Sutherland et al., 1975). Coulter et al. (1978) further showed that reliable identification of snake venoms could be achieved in a sandwich radioimmunoassay which used antisera of the same specificity for both the capture and conjugate antibody. The analogous EIA was made available for clinical use (Coulter et al., 1980), and although it established a need for such a test, its performance was too complex to gain widespread acceptance in the field. This was because the test kit comprised five separate conjugates, one for each of the five major antivenom groups and the kit user Was required to add a dilution of conjugate 1 to well 1, a dilution of conjugate 2 to well 2 and so on. A glass capillary format was devised (Chandler and Hurrell, 1982) to make the EIA suitable for routine field application and although difficult to produce, had good field acceptability (Hurrell and Chandler, 1982). However, the glass capillary format required the use of a single mixture of conjugates, and because of this it was necessary to affinity absorb the solid-phase antibodies to generate the required specificity. Subsequent extensive use in the field has highlighted several anomalies where the venom from less common snakes can be assigned incorrectly to one of the five groups. Most importantly, venoms from Red-bellied Black (Pseudechis porphyriacus) and Copperhead (Austrelaps superba) snakes reacted equally in both Tiger and Black categories but are most appropriately identified as Black and Tiger respectively. Additionally, venom of the Clarence River snake (Tropidechis carinatus) reacted with both Tiger and Taipan whereas it correctly belonged in the former group. These anomalies were unique to the new glass capillary format test (Coulter, unpublished results). The new format (COx, 1988) which was developed to resolve the above problems, permits the use of a different conjugate for each well by lyophilising conjugate within the appropriate well at the time of manufacture. As a result affinity absorption of the capture antibody is unnecessary and, because a simultaneous assay results, the steps normally required for conjugate incubation and washing are eliminated from the assay procedure.
Materials and methods
Antisera to snake venoms Antibodies to the venoms of the five snakes of greatest clinical importance in Australia, Brown ( Pseudonaja textilis), Death Adder ( Acanthophis antarcticus), King Brown (Pseudechis australis), Taipan (Oxyuranus scutellatus) and Tiger (Notechis scutatus), were produced in rabbits. These venoms and certain others were obtained from the Australian Reptile Park, Gosford, Australia and Mr. C. Tanner, Cooktown, Australia. Rabbits were injected with graded doses of venom from 10 /~g to 2 mg at 2-weekly intervals. The first four doses were in Freund's complete adjuvant (Commonwealth Serum Laboratories Limited (CSL), Parkville, Australia) and delivered intramuscularly deep into the hind or foreleg. Subsequent doses were delivered subcutaneously in Freund's incomplete adjuvant (CSL). Sera from test bleeds taken during the immunization course and large bleeds taken 1 week after the final dose were tested by gel diffusion. Selected rabbits were boosted and bled at six week intervals. IgG was purified from rabbit antisera by protein A chromatography according to the method of Goding (1976).
Preparation of conjugates Preparations of IgG were labelled with horseradish peroxidase according to the procedure of Wilson and Nakane (1978). At completion of the reaction conjugates were diluted to 1 mg/ml in a suitable conjugate stabilising buffer and held at 4°C.
Preparation of plates Polystyrene microtitre plates with 8 well removable strips (MaxiSorp, Nunc, Denmark) were used. Coating antibodies were diluted to 2/zg/ml in 0.05 M carbonate buffer, pH 9.6, and 100/~i added to each well of the 96 well plate. Following incubation overnight at 4°C, unbound antibodies were removed by aspiration and unreacted sites blocked by incubation with 150 /~! of 1 mg/mi casein in PBS Tween (0.01 M phosphate-buffered saline, pH 7.2 (PBS), containing 0.05% (v/v) Tween 20) at 20°C for 1 h. The blocking solution was then aspirated and replaced with a 5% (w/v)
215 solution of lactose in water for 5 min at 20°C in order to stabilise bound antibodies. After aspiration, 10/~1 of conjugate in stabilising buffer containing 0.15% (w/v) alphazurine A (an inert blue dye) were added. Plates were immediately transferred to the shelves of a freeze drier and lyophilised with secondary drying over P205 . Individual 8-well strips were capped, sealed in laminated foil pouches containing a sachet of silica gel and held at 4°C unless required for stability trials.
Experimental assay procedures Sequential and simultaneous assay procedures were compared using plates prepared as described above but vacuum dried at room temperature without the addition of conjugate. Venom samples were diluted in Yellow Diluent, a PBS Tween formulation containing 10 m g / m l casein and 40 /xg/ml tartrazine (an inert yellow dye). For the sequential assay, 50 /~1 Yellow Diluent were added to each well followed by 50/.d of test sample. The contents were mixed and incubated at room temperature for the specified time. The wells were then washed seven times with PBS Tween and tapped dry. Diluted conjugate (100 /~1) was added to each well and incubated and washed as before. Finally, 50 p.I each of peroxide solution (2 × ) and chromogen solution (2 × ) (tetramethylbenzidine; Bos et al., 1981) were added to each well. The contents of the wells were gently mixed and after 10 min the reaction was terminated by addition of 50 /~1 0.5 M H2SO 4. Absorbance values were read at 450 nm. For the simultaneous assay, 50 p.! of test sample and 50 p.l of conjugate at twice the concentration used above were added to each well, gently mixed and incubated at room temperature for the specified time. The wells were then washed and peroxide and chromogen solutions added as described above.
Kit components and performance The kits comprised an eight-well microtitre strip containing lyophilised conjugate and blue dye, Yellow Diluent, chromogen solution (2 x concentrate) and peroxide solution (2 x concentrate). Addition of the test sample in sample diluent to the microtitre wells reconstituted the
conjugate which could be visualised by the operater as the generation of a uniform green colour on mixing. Test sample was added to seven wells of an eight-well strip. The first well contained capture antibody and conjugate to Tiger snake venom (TSV) and similarly the next four wells contained reagents to identify venoms from Brown snake, Black snake, Death Adder and Taipan. The sixth well was a negative control with normal rabbit lgG as capture and an antioTSV conjugate. The seventh well was a positive control with normal rabbit lgG as capture and a sheep antirabbit lgG conjugate. The final well was blank (neither capture antibody nor conjugate) and could be used to orientate the strip. Following a 10 min incubation with sample at room temperature, the wells were washed at least seven times under running tap water. Then one drop each of chromogen solution and peroxide solution were added to each well and gently mixed. Wells were observed for the development of a blue colour, which occurred within 10 min for positive test samples and the positive control. The negative control well should remain colourless.
Stability trials Individually sealed strips were held at -70°C, 4°C, 18°C and 37°C. At intervals, pouches were allowed to equilibrate to room temperature, opened and tested. Results were calculated as the percentage drop in absorbance, assuming a 100% retention of activity at -70°C.
Results
Preliminary experiments were performed to compare the sensitivity of venom detection in sequential and simultaneous assay format. An incubation time of 10 min was used for the simultaneous assay and both 2 x 5 rain and 2 x 10 min incubations for the sequential assay. The results at various levels of TSV from 1.25 to 80 n g / m l are shown in Table I. It can be seen that for a given assay time, i.e., 1 x 10 min simultaneous or 2 x 5 rain sequential, the simultaneous assay was more sensitive at all points of the clinically rele-
216 TABLE I COMPARISON OF SIMULTANEOUS AND SEQUENTIAL SANDWICH EIA FORMATS ON SNAKE VENOM ASSAY SENSITIVITY Assay format
Concentration of Tiger Snake Venom (TSV) (ng/ml) 0 1.25 2.5 5 10
20
40
80
Simultaneous (1 × 10 min) Sequential (2×5 rain) Sequential (2 x I0 rain)
0.067 a 0.061 0.062
1.145 0 ~t,:5 0.7t"9
1.684 0.627 1.329
2.360 1.033 2.078
0.152 0.077 0.098
0.236 0.098 0.141
0.392 0.135 0.286
0.748 0.202 0.493
a Absorbance reading at 450 nm after addition of 0.5 M H2SO4.
v a n t r a n g e o f v e n o m c o n c e n t r a t i o n s a n d easily able to d e t e c t 2.5 n g / m l v e n o m . E x p e r i m e n t s were n e x t p e r f o r m e d to d e t e r m i n e t h e o p t i m a l v o l u m e o f c o n j u g a t e to lyophilise in e a c h well a n d to e s t i m a t e t h e loss o f activity d u r i n g lyophilisation. V o l u m e s o f 5, 10 a n d 20/~1 o f c o n j u g a t e p e r well were investigated, e a c h at c o n c e n t r a t i o n s covering t h e r a n g e f r o m 0.2 to 2.0 / t g c o n j u g a t e p e r well. T h e pellet r e s u l t i n g f r o m lyophilisation o f 20 /.d w a s f o u n d to b e insufficiently stable to i m p a c t w h e r e a s t h a t r e s u l t i n g f r o m 5 a n d 1 0 / t l w a s u n a f f e c t e d by jolting well in excess o f t h a t n o r m a l l y e x p e c t e d to occur. A 10 /~! d i s p e n s e d v o l u m e at 0 . 2 - 0 . 4 / ~ g c o n j u g a t e p e r
well (i.e., 2 0 - 4 0 p g / m l ) was finally c h o s e n as t h e o p t i m u m condition. L o s s e s o f a r o u n d 2 0 % o f c o n j u g a t e activity w e r e o b s e r v e d d u r i n g lyophilisation. T o d e t e r m i n e t h e sensitivity a n d specificity o f t h e assays, v e n o m s f r o m t h e five c o m m o n s n a k e s w e r e d i l u t e d to 10 a n d 40 n g / m l in d i l u e n t b u f f e r a n d t e s t e d as p e r instructions. T h e r e s u l t s o f o n e typical e x p e r i m e n t (Table II) s h o w e d t h a t all five v e n o m s w e r e unequivocally identified at 10 n g / m l (a distinct b l u e c o l o u r c o r r e s p o n d i n g to a n abs o r b a n c e r e a d i n g g r e a t e r t h a n 0.2) a n d t h a t cross-reactions, t h o u g h p r e s e n t , were c o n s i d e r ably w e a k e r t h a n specific reactions. Additionally,
TABLE II REACTION OF 5 COMMON AND 7 LESS COMMON VENOMS IN THE SNAKE VENOM DETECTION TEST Test venom
Tiger Brown King Brown Death Adder Taipan Clarence River Copperhead Palehead Dugite Gwardar Papuan Black Red-bellied Black
Concentration ng/ml
Tiger
Specificity of test well Brown
Black
Death Adder
Taipan
Neg.
Dos.
10 40 10 40 10 40 10 40 10 40 40 40 40 40 40 40 40
0.65 a 1.95 0.10 0.13 0.24 0.57 0.16 0.25 0.19 0.40 1.08 0.44 0.28 0.12 0.12 0.20 0.21
0.20 0.20 0.38 0.91 0.17 0.27 0.15 0.19 0.18 0.24 0.28 0.18 0.15 0.48 0.45 0.14 0.13
0.13 0.18 0.09 0.14 0.47 1.38 0.11 0.13 0.13 0.12 0.15 0.28 0.20 0.10 0.10 0.61 0.34
0.12 0.20 0.08 0.11 0.15 0.34 0.40 0.92 0.11 0.16 0.12 0.25 0.16 0.10 0.09 0.21 0.22
0.14 0.24 0.12 0.20 0.15 0.36 0.12 0.23 1.12 2.32 0.23 0.23 0.17 0.13 0.13 0.17 0.17
0.10 0.09 0.09 0.15 0.09 0.20 0.09 0.12 0.11 0.13 0.09 0.08 0.09 0.11 0.11 0.08 0.08
2.06 2.21 2.05 2.16 2,02 2.39 1.91 2.30 1.57 2.09 1.87 2.06 !.96 1.92 1.94 1.99 2.16
a Absorbance reading at 450 nm after addition of 0.5 M H2SO4.
217 seven less common venoms (listed in Table II) were tested at 40 ng/ml (a concentration which approximates to that of a bite-site sample) and the venom to which they bore the closest antigenic resemblance was determined. In six of these seven cascs, this indicated the most appropriate antivenom for treatment of bites. In the other case, Red-bellied Black was grouped correctly as a Black snake (it is genus Pseudechis as are other common members of this group), even though its bite is more appropriately treated with TSV antivenom (Sutherland, 1990). The stability of strips stored at 4°C, 18°C and 37°C was estimated at intervals from 1 to 40 weeks and compared with results obtained for strips stored at - 7 0 ° C. Wells were reacted with a low level (2.5-5 ng/ml) and a high level (40 ng/ml) of TSV and the results calculated as the percentage absorbance at the stated temperature relative to the - 70 ° C wells. Strips stored at 4°C lost around 15% of their activity after 40 weeks relative to strips stored at -70°C and were still able to detect reliably 2.5 ng/ml venom. Accelerated stability trials showed that strips retained 75% of their activity after 40 weeks at 18°C and 31% of their activity after 40 weeks at 37°C.
Discussion An essential requirement for the novel assay described in this paper is that the antibodies selected for the kit development must be able to function in a simultaneous assay format. The complex nature of the various antigens involved in the snake venom detection assay and the need to use polyclonal rather than monoclonal antibodies made the expected outcome uncertain. The results (Table I) show that a single incubation step of 10 rain in a simultaneous assay results in a more sensitive assay than two incubation steps, each of 5 or 10 min in a sequential assay. Thus, the simultaneous assay was both quicker and more sensitive. Similar benefits have been observed by us when developing assays for canine parvovirus in dog faeces (Drane et al., unpublished observations), human interferon-~, and hu-
man blood grouping (Isabel Roberts, personal communication). The observation that the simultaneous assay was both quicker and more sensitive can possibly be explained by a build-up of antigen-conjugate complexes bound to the solid phase in a simultaneous assay. Such multi-layer complexes cannot form in a sequential assay. The other consideration of note in developing this new assay concept was to iyophilise the conjugate within the well so that it did not interfere with the stability of the capture antibody, retained the stability of lyophilised enzyme conjugates and could not readily be dislodged from the well by physical shock. None of these issues in reality caused a problem. Typical losses o[ 20% of conjugate activity during drying were compensated for by an appropriate adjustment in conjugate strength. Volumes of 5 and 10/zi resulted in pellets which adhered satisfactorily to the surface of the wells, and the addition and lyophilisation of conjugate did not interfere with the activity or stability of the capture antibody. This was reflected in the excellent stability observed for the kit. To permit calculation of stability of the snake venom detection kit, it was decided to use as the reference wells stored at -70°C after lyophilisation. In this way, day to day variations due to temperature, sample preparation and other assay variables including stability of other reagents were removed from the calculation. The possibility of an initial sharp drop of activity in reference wells as a result of the transition from 4°C to -70°C was excluded by initial stability data which showed equal sensitivity for the -70°C and 4°C wells. The results suggest a useful reagent life in excess of 2 years. Extensive field evaluation of the snake venom detection kit for both veterinary and human application is in progress. Preliminary evaluations again show the new kit format to be simpler to use and more sensitive and specific. This increased specificity is particularly demonstrated by the ability of the test to identify correctly the most appropriate antivenom for treatment of the envenomed patient following bites by a number of less common snakes (Table II). Bites by these snakes have led to some confusion in interpretation of test results using the glass capillary assay kit.
218 T h e n e w t e c h n o l o g y d e s c r i b e d h e r e c a n have definite a d v a n t a g e s e v e n in a simple diagnostic test s i t u a t i o n s u c h as d e t e c t i o n o f specific virus in a faecal s a m p l e . However, in m o r e c o m p l e x test situations, especially w h e r e a biological s p e c i m e n is to be t e s t e d for t h e p r e s e n c e o f a n u m b e r o f possible analytes, for e x a m p l e t h e identification o f s n a k e v e n o m , t h e test c o n c e p t offers m a j o r a d v a n t a g e s in a d d i t i o n to e a s e a n d s p e e d o f perf o r m a n c e . M o s t importantly, a n t i s e r a for t h e diff e r e n t a n a l y t e s c a n b e derived f r o m d i f f e r e n t species b e c a u s e t h e n o r m a l p r o b l e m s a s s o c i a t e d with a m i x e d s p e c i e s c o n j u g a t e a r e avoided. F u r t h e r m o r e , a n y blocking p r o c e d u r e w h i c h m a y b e n e c e s s a r y for a n y specific r e a c t i o n c a n be conf i n e d to t h a t well by inclusion in t h e c o n j u g a t e stabilising b u f f e r for t h a t c o n j u g a t e . T h e relative sensitivity a n d specificity o f e a c h well c a n also b e a d j u s t e d b o t h by variation in t h e a n t i b o d y concentration of the capture and the conjugate phase. Finally, only r e l e v a n t c o n j u g a t e is p r e s e n t in e a c h well. A s a result, c o n j u g a t e cost is less a n d , m o r e importantly, b a c k g r o u n d reactivity w h i c h is largely i n f l u e n c e d by t h e a m o u n t o f c o n j u g a t e p r e s e n t , is significantly r e d u c e d . T h e a d v a n t a g e s o f t h e n e w r a p i d assay f o r m a t d e s c r i b e d h e r e s u g g e s t f u r t h e r laboratory a n d field applications. O f m a j o r interest a r e solid p h a s e blood g r o u p serology, m e a t s p e c i e s identification a n d r a p i d t e s t s for identification o f respiratory a n d intestinal p a t h o g e n s . D e v e l o p m e n t o f t h e s e t e s t s is u n d e r w a y .
References Bos, E.S., Van der Doelen, A.A., Van Rooy, N. and Schuurs, A.H.W.M. (1981) 3,Y,5,5'-Tetramethylbenzidine as an
Ames test negative chromogen for horseradish peroxidase in enzyme-immunoassay. J. Immunnassay2, 187. Chandler, H.M. and Hurrell, J.G.R. (1982) A new enzyme immunoassaysystem suitable for field use and its application in a snake venom detection kit. Clin. Chim. Acta 121, 225. Coulter, A.R., Sutherland, S.K. and Broad, A.J. (1974) Assay of snake venoms in tissue fluids. J. ImmunoL Methods 4, 297. Coulter, A.R.. Cox, J.C., Sutherland, S.K. and Waddell, CJ. (1978) A new solid-phase sandwich radioimmunoassay and its application to the detection of snake venom. J. ImmunoL Methods 23, 241. Coulter, A.R., Harris, R.D. and Sutherland, S.K. (1980) En~ m e immunoassay for the rapid clinical identification of snake venom. Med. J. Aust. i, 433. Cox, J.C. (1988) Improved immunoassay. International Patent Application no. PCT/AU89/00503. Goding, .I.W. (1976) Conjugation of antibodies with fluorochromes: Modifications to the standard methods. J. Immunol. Methods 13, 215. Hurrell, J.G.R. and Chandler, H.M. (1982) Capillary enzyme immunoassayfield kits for the detection of snake venom in clinical specimens. A review of two years use. MOd. J. Aust. 2, 236. Sutherland, S.K. (1983) Australian Animal Toxins. The Creatures, Their Toxins and Care of the Poisoned Patient. Oxford University Press, Melbourne. Sutherland, S.K. (1990) Treatment of snake bite. Aust. Fam. Physician 19, 21. Sntherland, S.K., Coulter, A.R., Broad, AJ., Hilton, J.M.N. and Lane, L.H.D. (1975) Human snake bite victims: The successful detection of circulating snake venom by radioimmunoassay. Med. J. Aust. 1, 27. Tijssen, P. (1985) In: R.H. Burden and P.H. Van Knippenberg (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, Vol 15, Practice and Theor~ of Enzyme Immunoassays. Elsevier, Amsterdam, p. 297. Wilson, M.B. and Nakane, P.K. (1978) Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In: W. Knapp, K. Holubar and G. Wick (Eds.), Immunofluorescence and Related Staining Techniques. Elsevier, Amsterdam, p. 215.
