Veterinary Parasitology, 42 (1992) 101-110 Elsevier Science Publishers B.V., Amsterdam
101
Enhanced chemiluminescent enzyme immunoassay for the detection of trichinellosis antibodies in pigs* R o n a l d C. K o a n d M a b e l H.F. Yeung Department of Zoology, University of Hong Kong, Hong Kong (Accepted 18 September 1991 )
ABSTRACT Ko, R.C. and Yeung, M.H.F., 1992. Enhanced chemiluminescent enzyme immunoassay for the detection of trichinellosis antibodies in pigs. Vet. Parasitol., 42:101-110. A modified enhanced chemiluminescent enzyme assay (ECIA) was developed for mass screening of pigs for triehinellosis antibodies in abattoirs. Using Bionectics beads as solid support, the assay time could be reduced to 45 min. Optimal conditions for washing, blocking, incubation, concentration of serum, antigens and conjugates as well as timing of film exposure were determined. The sensitivity and specificity of the assay were found to be comparable to those of the triple antibody-IgG ELISA. The assay was tested in an abattoir and its efficacy was found to be satisfactory. However, the major disadvantage of the assay is the high cost of magnetic beads.
INTRODUCTION E n h a n c e d c h e m i l u m i n e s c e n t e n z y m e i m m u n o a s s a y ( E C I A ) is a r e c e n t l y d e v e l o p e d a s s a y w h i c h is b a s e d o n the e m i s s i o n o f light d u e to the r e a c t i o n o f a n e n z y m e c o n j u g a t e d a n t i b o d y w i t h a s u b s t r a t e ( l u m i n o l ) in the p r e s e n c e o f an o x i d a n t ( h y d r o g e n p e r o x i d e ) . P a r a - i o d o p h e n o l , firefly, a n d p a r a - p h e n y l p h e n o l are s o m e o f t h e e n h a n c e r s w h i c h c a n i n c r e a s e t h e i n t e n s i t y o f l u m i n e s c e n c e u p to 2500 × . T h e results c a n be r e c o r d e d o n f i l m b y a m o d i f i e d P o l a r o i d c a m e r a b a c k or a l u m i n o m e t e r ( T h o r p e et al., 1985; K r i c k a a n d T h o r p e , 1986a,b). A l t h o u g h E C I A h a s b e e n a p p l i e d to a s s a y h o r m o n e s ( W e e r a s e k e r a et al., 1983; T h o r p e et al., 1984 ), h e p a t i t i s B surface antigen ( S c h r o e d e r et al., 1981 ), r u b e l l a a n t i b o d i e s ( T h o r p e et al., 1984) etc., it h a s s e l d o m b e e n u s e d for t h e s e r o d i a g n o s i s o f p a r a s i t i c diseases. A c c o r d i n g to K r i c k a a n d T h o r p e ( 1 9 8 6 a ) , *Part of this paper was presented at the 26th Annual Scientific Seminar of the Malaysian Society of Parasitology and Tropical Medicine held in Penang, 22-24 February 1990.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
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R.C. KO AND M.H.F. YEUNG
the assay has been applied to detect antigens of Plasmodium falciparum and Schistosoma mansoni. As ECIA does not require sophisticated equipment and can be performed easily under field conditions, it has great potential as a routine screening method in abattoirs. Therefore, a study was undertaken to develop a faster ECIA by the employment of magnetic beads as solid support. The specificity and sensitivity of the test were examined in light of its ability to detect Trichinella spiralis antibodies in Chinese pigs which are invariably infected with a wide range of indigenous helminths. The optimal conditions under which the assay can operate most effectively were also studied. Excretory/secretory (ES) antigens obtained by culturing the muscle larvae of Trichinella spiralis have been demonstrated to be highly specific for the immunodiagnosis of swine trichinellosis by various types of traditional enzyme-linked immunoassays (Gamble et al., 1983, 1988; Ko and Yeung, 1989a,b). To determine the specificity of ECIA, both ES and crude somatic antigens were used in the present study. MATERIALS AND METHODS
The following parameters which could affect ECIA were studied: types of solid support, concentration of antigens and antibodies, incubation time, washing effect, blocking effect, types of antigens and period of film exposure. The assay was applied to screen serum samples from Chinese pigs which were also tested with the triple antibody-IgG ELISA. A trial run was carried out in an abattoir to determine the efficacy of the assay.
Animals and antiserum The strain of Trichinella spiralis used was originally isolated from a pig in Ontario, Canada in 1967 and has since been maintained alternately in Wistar rats and ICR mice in the laboratory. Positive control serum samples were obtained by the oral infection of five weaned piglets and six albino New Zealand rabbits with 2000 and 10 000 larvae each, respectively. The piglets and rabbits were bled and killed at about 1.5 months after infection. Hyperimmune serum against the c o m m o n parasites of Chinese pigs (Trichuris suis, Gnathostoma hispidum, Metastrongylus apri, Ascaris suum and Cysticerus cellulosae) were produced in nine piglets and l 1 rabbits by three intramuscular injections at weekly intervals using crude antigens and Freund's complete and incomplete adjuvants. Serum samples from seven uninfected piglets and six rabbits served as negative controls. The animals were killed humanely by an overdose of ketamine hydrochloride (Ketalar, Parke Davis, NSW, Australia) and chloromethyl imidazo benzodiazepina (Dormicum, Roche, Basle, Switzerland).
ENZYME ASSAY TO DETECT TRICHINELLOSIS ANTIBODIES IN PIGS
103
Antigens Crude antigens of Trichinella spiralis and heterologous species were prepared by sonication as described by Chan and Ko (1988 ). Excretory/secretory (ES) antigens were recovered from RPMI 1640 culture medium in which L1 larvae had been maintained for 24 h following the method of Ko and Yeung (1989a). The samples were concentrated by a sample concentrator (SpeedVac) or by ultrafiltration (Spectrum, CA, USA, 5 kDa molecular cut-off filter). After concentration, the antigens were desalted by gel filtration using Bio-Gel P6DG (Bio-Rad, CA, USA). Protein concentrations were determined by a Protein Assay Kit (Bio-Rad) and the absorbance of the samples was read at 595 nm with a spectrophotometer (Milton Roy, Stone, UK).
Luminometer A camera luminometer, which was based on contact exposure of film, was constructed according to Kricka and Thorpe (1986b), with some modifications. The original flexible shutter of a Polaroid Land camera back (model 405 ) was replaced by a metallic plate. A "mask", which could accommodate an activated flat-bottom polyvinyl (PVC) microtitration plate (Flow Laboratories, CA, USA) and prevent cross-talk between the wells, was made from an aluminium block by the Engineering Workshop, University of Hong Kong (Fig. 1 ). Upon the opening of the shutter, the mask together with the microtitration plate will fall directly onto the film pack. To ensure that the mask
C Fig. 1. Camera luminometer showing the metallic mask (M) which can accommodate a poly~ vinyl microtitration plate, a shutter (S), the Polaroid camera back (C) and lid (L) of the assembly.
104
R.C. KO AND M.H.F. YEUNG
would always contact the film at the same location whenever the shutter was opened, a guiding pin was drilled into the camera back. A slanting edge of about 45 ° was cut at one end of the mask so that when the shutter was closed, it could be lifted from the film. The PVC plate was cut into 9 × 7 wells format to fit the standard size of film. A lid, which was made of dark plexiglas, provided a light-proof enclosure for the assembly. Polaroid instant film T667 (ASA 3000) was used to record the luminescence. ECIA
To determine the effects of different solid support on the speed of the assay, ECIA was performed using PVC plates alone and with the employment of magnetic beads. Tosylactivated micro-Dynabeads (Dyno, Skoyen, Norway, M-450) (4.5/tm diameter) and polycarbonate coated ferrous metal Bionectics beads (Organon, NC, USA) (4 m m diameter) were tested. An ordinary bar magnet was used to immobilise the Dynabeads during removal of fluid from the wells. A tubular beads dispenser (Organon) was used to transfer Bionectics beads to a new plate. For the coating of Dynabeads, the antigen was first diluted in 0.5 M borate solution (pH 9.5) to a concentration of 150/zg protein m l - 1. An equal volume of undiluted suspension of beads was added to the antigen solution to achieve an antigen:bead ratio of 75/~g per 15 rag. The mixture was then incubated at room temperature for 24 h by slow end-over-end rotation. Afterwards, the beads were collected and washed in phosphate buffered saline (first three times for 10 min each, then once for 30 min and finally overnight). The washings were carried out at room temperature except for the last one which was at 4 ° C. For the coating of Bionectics beads, one bead was first dispensed into each well of the microtitration plate; 150 ~1 of antigen diluted in coating buffer (0.1 M carbonate-bicarbonate buffer at pH 9.6) was then added. The covered plate was incubated with gentle shaking on a shaker (MTS 4, IKA, Stanfen, Germany) at 4 °C overnight. The coating of PVC with antigens followed the same procedures as described previously for polystyrene plates (Ko and Yeung, 1989a). The remaining procedures were performed according to Kricka and Thorpe (1986b) with modifications. After the coating of antigens, 3% bovine serum albumin (BSA) in Tris-buffered saline was added to the plates or beads for 5 min to block the free binding sites. Following blocking and between all subsequent steps, the plates or beads were washed using Tween 20-Tris-buffered saline for 3 min with gentle shaking. The optimal conditions for the concentration of antigens, antiserum and horseradish peroxidase (HRPO)-IgG conjugate (Cappel, NC, USA, heavy and light chain specific) were determined by checkerboard titrations. The test serum was diluted 1:50 ( v / v ) while the rabbit anti-swine IgG HRPO-
ENZYME ASSAY TO DETECT TR1CH1NELLOSIS ANTIBODIES IN PIGS
| 05
conjugate was diluted 1:2000 ( v / v ) . To determine the optimal time, each incubation step was carried out both at room temperature or 37 °C for various periods, i.e. 15, 30, 60 or 120 rain. The substrate solution was prepared by adding 1 ml of the enhancer, 4iodophenol (Aldrich Chemical Co., WI, USA) (40.9 mM in DMSO), to 100 ml of a mixture ofluminol (1.25 mM, Sigma Chemical Co., St. Louis, MO) and hydrogen peroxide (2.7 m M ) in Tris buffer (0.1 M, pH 8.5 ).
ELISA The triple antibody-IgG ELISA was performed according to Ko and Yeung ( 1989 ) and Gamble et al. ( 1983 ). First, 0.2/~g ES or 0.4/tg crude antigens in 100/~1 per well were used to coat flat-bottomed polystyrene microtitration plates (Linbro, Flow Laboratories). The test serum was diluted 1 : 100 ( v / v ) . The goat anti-rabbit HRPO-IgG conjugate (Cappel) was diluted 1:30 000 ( v / v ) . The plates were washed by an automatic plate washer and read by ELISA reader (Ultrawash and MR 710, Dynatech, Channel Islands, UK). Absorbance values greater than five times the mean optical density (OD) of the normal pool were considered to be positive. RESULTS
The results of ECIA demonstrated that the ES antigens were more specific than the crude extracts which yielded more false positive results. Antigens were serially diluted from 0.2 to 3.2 ¢tg m l - 1. The optimal concentrations for ES and crude antigens were found to be 4/#g m l - 1 and 8 #g m l - 1. At higher concentrations, the contrast between the positives and negatives would diminish and at lower concentrations, false positives of heterologous serum samples would increase. For calibration, the positive control pig anti-TrichinelIa spiralis serum and normal pig serum were diluted serially from 1 : 10 to 1 : 200. For the former serum, when tested against various concentrations of antigen, a gradation of spot intensity could only be observed at 1:50 dilution which was taken as the optimum. False negatives started to appear at 1 : 100 dilution. To determine the optimal timing for the initiation of film exposure, films were exposed at 0, 2, 5 and 120 min after the addition of the substrate mixture. The best timing determined was at time 0. Exposure times varying from 10 s to 10 min were tested. A 10 s exposure was found to be optimal because at longer exposures, false positives involving anti-Trichuris suis, M. apri and Gnathostoma hispidum serum were recorded (Fig. 2). Dynabeads and Bionectics beads were found to serve as better solid support than PVC plate. When the beads were used, more spots indicating strong luminescence were recorded (Fig. 3).
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R.C. KOAND M.H.F. YEUNG
blank
(0.14-5)(0.190)+ (0.945)++ (o.181)T
(0.199)M (0.162)0 10s
60s
5rnin
Exposures Fig. 2. Effects of the timing of film exposure on ECIA results, showing the recording of more false positive reactions at longer exposures. Positive reactions are indicated by the presence of white spots. Antisera used in the different rows are (from top to bottom): blank, negative control, lightly infected positive control, heavily infected positive control, Trichuris suis (T), Metastrongylus apri (M) and Gnathostoma h ispidum (G). Numbers in brackets are the corresponding OD values obtained by triple antibody ELISA.
crude Ag
ES Ag PVC
beads -
+ +
-1
++
Fig. 3. Comparison of ECIA results when crude and excretory/secretory antigens were coated directly on polyvinyl plate or Bionectics beads, Note the presence of solid white spots when the latter were used. + + , heavily infected positive control antiserum; - , negative control.
T h e effects o f w a s h i n g a n d b l o c k i n g w i t h 1% BSA w e r e s h o w n in Figs. 4 ( a ) a n d 4 ( b ) . W i t h w a s h i n g a n d blocking, the s e n s i t i v i t y o f the H R P O - I g G conj u g a t e was i m p r o v e d a n d m o r e c o n s i s t e n t results w e r e o b t a i n e d . T h e reliability o f E C I A was t e s t e d b y s c r e e n i n g a b a t c h o f sera s a m p l e s f r o m 84 pigs o n e o f w h i c h was k n o w n to be n a t u r a l l y i n f e c t e d with Trichinella spiralis. T h e s a m e b a t c h o f sera was also t e s t e d b y the triple a n t i b o d y E L I S A a n d
ENZYME ASSAY TO DETECT TR1CHINELLOSIS ANTIBODIES IN PIGS (o) With btocking and washing steps
1:10
1:20
1:50
Pig anti Trichinetla~sEirali~s serum
1:10
107
(b) Without blocking and woshin~j steps
1:20
Normal pig serum
Fig. 4. Effects of blocking and washing on ECIA results using crude antigens (at protein concentrations of 8 and 16 #g m l - ~), various dilutions of antisera and rabbit anti-pig IgG horseradish peroxidase conjugate. The test was performed in duplicate. More consistent results are shown with blocking and washing. * 334(0-161} 308(0.103) 301(0.120) 1"(0.103) -5010.078) 335(0.160) 309(0.106) 302(0.121) M(0-137} Neat 336(0-175) 310(0.125l 303(0.17/4 G(0.081) ~,10(0.879} 337(0-139) 311(0.1331 304{0.1131 S(0-062) +,100(0328) 338(0.372) 312(0.117) 305(0.122) C(0.0?SI ~+200(0.301} 339(0.1581 313(0-148) 306(0.10t4 A(0.0~8} ~t,00(0.164) 3t,0(0.151) 31t,(0.132) 307(0.098) 50-*(0.451} ~800[0-116)
Fig. 5. Comparison of the results of ECIA and triple antibody ELISA in the screening of sera samples from 84 Chinese pigs for trichinellosis antibodies. The tests were carried out on pooled sera samples (each based on four animals). Each test was performed in duplicate. Numbers above the figure correspond to the wells of the polystyrene plate. *indicates sample number. Numbers in brackets are the corresponding mean OD values of samples tested by triple antibody ELISA. To determine the specificity of the assays, the following heterologous, antisera were also tested: Trichuris suis (T), Metastrongylus apri (M), Gnathostoma h ispidum (G), Stephanurus dentatus ( S ), Cysticercus cellulosae (C) and Angiostrongylus cantonensis ( A ). + +, various dilutions of the positive control serum; - , negative control serum. Positive ECIA results are shown only in sample No. 338 and positive control samples (undiluted, 1:50, 1:10 and 1 : 100 dilutions).
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R.C. KO AND M.H.F. YEUNG
the OD values obtained are given in brackets beside the sample number in Fig. 5. Both assays were capable of detecting the positive sample and there were no false positive reactions. To determine the practicability of this assay as a routine screening procedure, it was applied to test serum samples from 73 pigs at an abattoir. As the pigs were from a non-endemic region, all the samples yielded negative results. However, the assay was found suitable for use in the abattoir environment, provided all the chemicals and antigen coatings were prepared beforehand. DISCUSSION
The present study shows that ECIA has a similar specificity and sensitivity as the conventional triple antibody ELISA in the detection of trichinellosis antibodies in pig serum. Parameters which affect ELISA can also influence the working of ECIA. Owing to the fact that the results of ECIA can simply be recorded on a Polaroid film, this assay will be more practical than the conventional ELISA or other colorimetric assays to be used in abattoirs, especially those with primitive conditions. With the employment of magnetic beads and all the preparative steps performed previously, ECIA can be completed as fast as 45 min. As the end point is reached very rapidly, the results can be recorded immediately after the addition of the substrate. In conventional ELISA, the colour development step usually takes about 15-30 min. Therefore, the rapidity of our system can meet the requirement of abattoirs where operation time is critical because of fast turnover rates and a substantial daily load. For example, in the abattoirs of Hong Kong, 2000-3000 pigs are commonly processed within one morning. Any immunoassay which requires more than 1 h cannot be accepted as a routine screening method. Owing to the preference of Chinese for fresh unfrozen meat, the slaughtered pigs must be delivered immediately to the retail markets. The major advantages of using magnetic beads are the dispersibility of the solid phase which can provide a relatively large surface area, fast binding rates and easy dispersal. If Dynabeads are used, the beads can be localised by magnet, thus avoiding the time consuming washing and colour development steps as in conventional ELISA. In the case of Bionectic beads, all the reagents can be prepared and dispensed into different microtitration plates beforehand. At the end of each incubation period, the beads are transferred directly from one plate into another, thus greatly reducing the processing time. The substrate reaction can also be terminated abruptly by the removal of beads. However, the great disadvantage is the current high cost of the beads. As the cost per test is dependent on the amount of antigens required, at an optimal protein: bead ratio of 150/~g per 2 ml and an antigen concentration of 0.4 /~g per well, the cost for Dynabeads is about US 30 cents per test (based on
ENZYME ASSAY TO DETECT TRICHINELLOSIS ANTIBODIES IN PIGS
109
local prices). For Bionectics beads, the cost is about US 25 cents per test. This, together with the costs for chemicals, PVC plates and instant film, will make this assay too expensive for routine large-scale screening, especially in developing countries. To improve the cost effectiveness, a cheaper substitute for the magnetic beads has to be developed. Another disadvantage is that the camera luminometer cannot provide quantification of the results as required in some research. The size and degree of whiteness of the exposed spots on the Polaroid film are directly dependent on the intensity of luminescence. But these can only be assessed visually and subjectively unless a densitometer or neutral density filters are used (Kricka and Thorpe, 1986b). However, if the Polaroid prints are copied to a black and white film, the positives will appear as dark spots on the negative. The intensity of the spots can then be quantified by a densitometer. Alternatively, luminescence can be measured by a photomultiplier or photodiode-based microplate reader. Unfortunately, the few readers which are available in the market are very expensive and are unsuitable for use in abattoirs or under field conditions. Kricka and Thorpe (1986a) used a high speed Polaroid film with 20 000 ASA (Type 612 ). However, for the present study, we used a slower T667 film with only 3000 ASA because higher speed films are not available locally. Nevertheless, both the sensitivity and contrast of the T667 film were found to be satisfactory. In fact, a slower film which is not only substantially cheaper, with its low contrast, can provide better results than T612, the contrast of which is too high to provide a graded response in image density. As the diameter of the exposed spot is dependent on the distance between the source of luminescence and the film, to achieve the m a x i m u m spot size when the shutter is opened, the bottom of the wells should be as close to the film as possible. This means that the height of the mask which houses the microplate should not be any more than a few millimetres larger than that of the plate. ACKNOWLEDGEMENT
This project was generously supported by the Croucher Foundation, Hong Kong.
REFERENCES Chan, S.W. and Ko, R.C., 1988. Comparison between standard ELISA and dot-ELISA for serodiagnosis of human trichinosis. Trans. R. Soc. Trop. Hyg., 82: 892-894. Gamble, H.R., Anderson, W.R., Graham, C.E. and Murrell; K.D., 1983. Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory-secretory antigen. Vet. Parasitol., 13:349-361.
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Gamble, H.R., Rapic, D., Marinculic, A. and Murrell, K.D., 1988. Influence cultivation conditions on specificity of excretory-secretory antigens for the immunodiagnosis of trichinellosis. Vet. Parasitol., 30: 131-137. Ko, R.C. and Yeung, M.H.F., 1989a. Specificity of ES antigens in detection of Trichinella spiralis antibodies in Chinese pigs. Trop. Biomed., 6:99-111. Ko, R.C. and Yeung, M.H.F., 1989b. Trichinella spiralis ES antigens: screening of Chinese pigs imported into Hong Kong for trichinellosis (abstract). Proceedings of the 64th Meeting of the American Society of Parasitology, Vancouver, Canada, p. 65. Kricka, L.J. and Thorpe, G.H.G., 1986a. Enhanced chemiluminescent enzyme immunoassays. Parasitol. Today, 2: 123-125. Kricka, L.J. and Thorpe, G.H.G., 1986b. Photographic detection ofchemiluminescent and bioluminescent reactions. Methods Enzymol., 133: 404-420. Schroeder, H.R., Hines, C.M., Obsborn, D.D., Moore, R.P., Hurtle, R.L., Wogoman, F.F., Rogers, R.W. and Vogelhut, P.O., 1981. Immunochemiluminometric assay for hepatitis B surface antigen. Clin. Chem., 27: 1378-1384. Thorpe, G.H.G., Haggart, R., Kricka, L.J. and Whitehead, T.P., 1984. Enhanced luminescent enzyme immunoassays for rubella antibody, immunoglobulin E and digoxin. Biochem. Biophys. Res. Commun., 119: 481-487. Thorpe, G.H.G., Kricka, L.J., Moseley, S.B. and Whitehead, T.P., 1985. Phenols as enhancers of the chemiluminescent horseradish peroxidase luminol-hydrogen perioxide reaction: application in luminescence monitored enzyme immunoassays. Clin. Chem., 31:1335-1341. Weerasekera, D.A., Kim, J.B., Barnard, G.J. and Collins, W.P., 1983. Measurement of serum thyroxine by solid-phase chemiluminescence immunoassay. Ann. Clin. Biochem., 20: 100104.
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Enhanced chemiluminescent enzyme immunoassay for the detection of trichinellosis antibodies in pigs* R o n a l d C. K o a n d M a b e l H.F. Yeung Department of Zoology, University of Hong Kong, Hong Kong (Accepted 18 September 1991 )
ABSTRACT Ko, R.C. and Yeung, M.H.F., 1992. Enhanced chemiluminescent enzyme immunoassay for the detection of trichinellosis antibodies in pigs. Vet. Parasitol., 42:101-110. A modified enhanced chemiluminescent enzyme assay (ECIA) was developed for mass screening of pigs for triehinellosis antibodies in abattoirs. Using Bionectics beads as solid support, the assay time could be reduced to 45 min. Optimal conditions for washing, blocking, incubation, concentration of serum, antigens and conjugates as well as timing of film exposure were determined. The sensitivity and specificity of the assay were found to be comparable to those of the triple antibody-IgG ELISA. The assay was tested in an abattoir and its efficacy was found to be satisfactory. However, the major disadvantage of the assay is the high cost of magnetic beads.
INTRODUCTION E n h a n c e d c h e m i l u m i n e s c e n t e n z y m e i m m u n o a s s a y ( E C I A ) is a r e c e n t l y d e v e l o p e d a s s a y w h i c h is b a s e d o n the e m i s s i o n o f light d u e to the r e a c t i o n o f a n e n z y m e c o n j u g a t e d a n t i b o d y w i t h a s u b s t r a t e ( l u m i n o l ) in the p r e s e n c e o f an o x i d a n t ( h y d r o g e n p e r o x i d e ) . P a r a - i o d o p h e n o l , firefly, a n d p a r a - p h e n y l p h e n o l are s o m e o f t h e e n h a n c e r s w h i c h c a n i n c r e a s e t h e i n t e n s i t y o f l u m i n e s c e n c e u p to 2500 × . T h e results c a n be r e c o r d e d o n f i l m b y a m o d i f i e d P o l a r o i d c a m e r a b a c k or a l u m i n o m e t e r ( T h o r p e et al., 1985; K r i c k a a n d T h o r p e , 1986a,b). A l t h o u g h E C I A h a s b e e n a p p l i e d to a s s a y h o r m o n e s ( W e e r a s e k e r a et al., 1983; T h o r p e et al., 1984 ), h e p a t i t i s B surface antigen ( S c h r o e d e r et al., 1981 ), r u b e l l a a n t i b o d i e s ( T h o r p e et al., 1984) etc., it h a s s e l d o m b e e n u s e d for t h e s e r o d i a g n o s i s o f p a r a s i t i c diseases. A c c o r d i n g to K r i c k a a n d T h o r p e ( 1 9 8 6 a ) , *Part of this paper was presented at the 26th Annual Scientific Seminar of the Malaysian Society of Parasitology and Tropical Medicine held in Penang, 22-24 February 1990.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
102
R.C. KO AND M.H.F. YEUNG
the assay has been applied to detect antigens of Plasmodium falciparum and Schistosoma mansoni. As ECIA does not require sophisticated equipment and can be performed easily under field conditions, it has great potential as a routine screening method in abattoirs. Therefore, a study was undertaken to develop a faster ECIA by the employment of magnetic beads as solid support. The specificity and sensitivity of the test were examined in light of its ability to detect Trichinella spiralis antibodies in Chinese pigs which are invariably infected with a wide range of indigenous helminths. The optimal conditions under which the assay can operate most effectively were also studied. Excretory/secretory (ES) antigens obtained by culturing the muscle larvae of Trichinella spiralis have been demonstrated to be highly specific for the immunodiagnosis of swine trichinellosis by various types of traditional enzyme-linked immunoassays (Gamble et al., 1983, 1988; Ko and Yeung, 1989a,b). To determine the specificity of ECIA, both ES and crude somatic antigens were used in the present study. MATERIALS AND METHODS
The following parameters which could affect ECIA were studied: types of solid support, concentration of antigens and antibodies, incubation time, washing effect, blocking effect, types of antigens and period of film exposure. The assay was applied to screen serum samples from Chinese pigs which were also tested with the triple antibody-IgG ELISA. A trial run was carried out in an abattoir to determine the efficacy of the assay.
Animals and antiserum The strain of Trichinella spiralis used was originally isolated from a pig in Ontario, Canada in 1967 and has since been maintained alternately in Wistar rats and ICR mice in the laboratory. Positive control serum samples were obtained by the oral infection of five weaned piglets and six albino New Zealand rabbits with 2000 and 10 000 larvae each, respectively. The piglets and rabbits were bled and killed at about 1.5 months after infection. Hyperimmune serum against the c o m m o n parasites of Chinese pigs (Trichuris suis, Gnathostoma hispidum, Metastrongylus apri, Ascaris suum and Cysticerus cellulosae) were produced in nine piglets and l 1 rabbits by three intramuscular injections at weekly intervals using crude antigens and Freund's complete and incomplete adjuvants. Serum samples from seven uninfected piglets and six rabbits served as negative controls. The animals were killed humanely by an overdose of ketamine hydrochloride (Ketalar, Parke Davis, NSW, Australia) and chloromethyl imidazo benzodiazepina (Dormicum, Roche, Basle, Switzerland).
ENZYME ASSAY TO DETECT TRICHINELLOSIS ANTIBODIES IN PIGS
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Antigens Crude antigens of Trichinella spiralis and heterologous species were prepared by sonication as described by Chan and Ko (1988 ). Excretory/secretory (ES) antigens were recovered from RPMI 1640 culture medium in which L1 larvae had been maintained for 24 h following the method of Ko and Yeung (1989a). The samples were concentrated by a sample concentrator (SpeedVac) or by ultrafiltration (Spectrum, CA, USA, 5 kDa molecular cut-off filter). After concentration, the antigens were desalted by gel filtration using Bio-Gel P6DG (Bio-Rad, CA, USA). Protein concentrations were determined by a Protein Assay Kit (Bio-Rad) and the absorbance of the samples was read at 595 nm with a spectrophotometer (Milton Roy, Stone, UK).
Luminometer A camera luminometer, which was based on contact exposure of film, was constructed according to Kricka and Thorpe (1986b), with some modifications. The original flexible shutter of a Polaroid Land camera back (model 405 ) was replaced by a metallic plate. A "mask", which could accommodate an activated flat-bottom polyvinyl (PVC) microtitration plate (Flow Laboratories, CA, USA) and prevent cross-talk between the wells, was made from an aluminium block by the Engineering Workshop, University of Hong Kong (Fig. 1 ). Upon the opening of the shutter, the mask together with the microtitration plate will fall directly onto the film pack. To ensure that the mask
C Fig. 1. Camera luminometer showing the metallic mask (M) which can accommodate a poly~ vinyl microtitration plate, a shutter (S), the Polaroid camera back (C) and lid (L) of the assembly.
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R.C. KO AND M.H.F. YEUNG
would always contact the film at the same location whenever the shutter was opened, a guiding pin was drilled into the camera back. A slanting edge of about 45 ° was cut at one end of the mask so that when the shutter was closed, it could be lifted from the film. The PVC plate was cut into 9 × 7 wells format to fit the standard size of film. A lid, which was made of dark plexiglas, provided a light-proof enclosure for the assembly. Polaroid instant film T667 (ASA 3000) was used to record the luminescence. ECIA
To determine the effects of different solid support on the speed of the assay, ECIA was performed using PVC plates alone and with the employment of magnetic beads. Tosylactivated micro-Dynabeads (Dyno, Skoyen, Norway, M-450) (4.5/tm diameter) and polycarbonate coated ferrous metal Bionectics beads (Organon, NC, USA) (4 m m diameter) were tested. An ordinary bar magnet was used to immobilise the Dynabeads during removal of fluid from the wells. A tubular beads dispenser (Organon) was used to transfer Bionectics beads to a new plate. For the coating of Dynabeads, the antigen was first diluted in 0.5 M borate solution (pH 9.5) to a concentration of 150/zg protein m l - 1. An equal volume of undiluted suspension of beads was added to the antigen solution to achieve an antigen:bead ratio of 75/~g per 15 rag. The mixture was then incubated at room temperature for 24 h by slow end-over-end rotation. Afterwards, the beads were collected and washed in phosphate buffered saline (first three times for 10 min each, then once for 30 min and finally overnight). The washings were carried out at room temperature except for the last one which was at 4 ° C. For the coating of Bionectics beads, one bead was first dispensed into each well of the microtitration plate; 150 ~1 of antigen diluted in coating buffer (0.1 M carbonate-bicarbonate buffer at pH 9.6) was then added. The covered plate was incubated with gentle shaking on a shaker (MTS 4, IKA, Stanfen, Germany) at 4 °C overnight. The coating of PVC with antigens followed the same procedures as described previously for polystyrene plates (Ko and Yeung, 1989a). The remaining procedures were performed according to Kricka and Thorpe (1986b) with modifications. After the coating of antigens, 3% bovine serum albumin (BSA) in Tris-buffered saline was added to the plates or beads for 5 min to block the free binding sites. Following blocking and between all subsequent steps, the plates or beads were washed using Tween 20-Tris-buffered saline for 3 min with gentle shaking. The optimal conditions for the concentration of antigens, antiserum and horseradish peroxidase (HRPO)-IgG conjugate (Cappel, NC, USA, heavy and light chain specific) were determined by checkerboard titrations. The test serum was diluted 1:50 ( v / v ) while the rabbit anti-swine IgG HRPO-
ENZYME ASSAY TO DETECT TR1CH1NELLOSIS ANTIBODIES IN PIGS
| 05
conjugate was diluted 1:2000 ( v / v ) . To determine the optimal time, each incubation step was carried out both at room temperature or 37 °C for various periods, i.e. 15, 30, 60 or 120 rain. The substrate solution was prepared by adding 1 ml of the enhancer, 4iodophenol (Aldrich Chemical Co., WI, USA) (40.9 mM in DMSO), to 100 ml of a mixture ofluminol (1.25 mM, Sigma Chemical Co., St. Louis, MO) and hydrogen peroxide (2.7 m M ) in Tris buffer (0.1 M, pH 8.5 ).
ELISA The triple antibody-IgG ELISA was performed according to Ko and Yeung ( 1989 ) and Gamble et al. ( 1983 ). First, 0.2/~g ES or 0.4/tg crude antigens in 100/~1 per well were used to coat flat-bottomed polystyrene microtitration plates (Linbro, Flow Laboratories). The test serum was diluted 1 : 100 ( v / v ) . The goat anti-rabbit HRPO-IgG conjugate (Cappel) was diluted 1:30 000 ( v / v ) . The plates were washed by an automatic plate washer and read by ELISA reader (Ultrawash and MR 710, Dynatech, Channel Islands, UK). Absorbance values greater than five times the mean optical density (OD) of the normal pool were considered to be positive. RESULTS
The results of ECIA demonstrated that the ES antigens were more specific than the crude extracts which yielded more false positive results. Antigens were serially diluted from 0.2 to 3.2 ¢tg m l - 1. The optimal concentrations for ES and crude antigens were found to be 4/#g m l - 1 and 8 #g m l - 1. At higher concentrations, the contrast between the positives and negatives would diminish and at lower concentrations, false positives of heterologous serum samples would increase. For calibration, the positive control pig anti-TrichinelIa spiralis serum and normal pig serum were diluted serially from 1 : 10 to 1 : 200. For the former serum, when tested against various concentrations of antigen, a gradation of spot intensity could only be observed at 1:50 dilution which was taken as the optimum. False negatives started to appear at 1 : 100 dilution. To determine the optimal timing for the initiation of film exposure, films were exposed at 0, 2, 5 and 120 min after the addition of the substrate mixture. The best timing determined was at time 0. Exposure times varying from 10 s to 10 min were tested. A 10 s exposure was found to be optimal because at longer exposures, false positives involving anti-Trichuris suis, M. apri and Gnathostoma hispidum serum were recorded (Fig. 2). Dynabeads and Bionectics beads were found to serve as better solid support than PVC plate. When the beads were used, more spots indicating strong luminescence were recorded (Fig. 3).
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R.C. KOAND M.H.F. YEUNG
blank
(0.14-5)(0.190)+ (0.945)++ (o.181)T
(0.199)M (0.162)0 10s
60s
5rnin
Exposures Fig. 2. Effects of the timing of film exposure on ECIA results, showing the recording of more false positive reactions at longer exposures. Positive reactions are indicated by the presence of white spots. Antisera used in the different rows are (from top to bottom): blank, negative control, lightly infected positive control, heavily infected positive control, Trichuris suis (T), Metastrongylus apri (M) and Gnathostoma h ispidum (G). Numbers in brackets are the corresponding OD values obtained by triple antibody ELISA.
crude Ag
ES Ag PVC
beads -
+ +
-1
++
Fig. 3. Comparison of ECIA results when crude and excretory/secretory antigens were coated directly on polyvinyl plate or Bionectics beads, Note the presence of solid white spots when the latter were used. + + , heavily infected positive control antiserum; - , negative control.
T h e effects o f w a s h i n g a n d b l o c k i n g w i t h 1% BSA w e r e s h o w n in Figs. 4 ( a ) a n d 4 ( b ) . W i t h w a s h i n g a n d blocking, the s e n s i t i v i t y o f the H R P O - I g G conj u g a t e was i m p r o v e d a n d m o r e c o n s i s t e n t results w e r e o b t a i n e d . T h e reliability o f E C I A was t e s t e d b y s c r e e n i n g a b a t c h o f sera s a m p l e s f r o m 84 pigs o n e o f w h i c h was k n o w n to be n a t u r a l l y i n f e c t e d with Trichinella spiralis. T h e s a m e b a t c h o f sera was also t e s t e d b y the triple a n t i b o d y E L I S A a n d
ENZYME ASSAY TO DETECT TR1CHINELLOSIS ANTIBODIES IN PIGS (o) With btocking and washing steps
1:10
1:20
1:50
Pig anti Trichinetla~sEirali~s serum
1:10
107
(b) Without blocking and woshin~j steps
1:20
Normal pig serum
Fig. 4. Effects of blocking and washing on ECIA results using crude antigens (at protein concentrations of 8 and 16 #g m l - ~), various dilutions of antisera and rabbit anti-pig IgG horseradish peroxidase conjugate. The test was performed in duplicate. More consistent results are shown with blocking and washing. * 334(0-161} 308(0.103) 301(0.120) 1"(0.103) -5010.078) 335(0.160) 309(0.106) 302(0.121) M(0-137} Neat 336(0-175) 310(0.125l 303(0.17/4 G(0.081) ~,10(0.879} 337(0-139) 311(0.1331 304{0.1131 S(0-062) +,100(0328) 338(0.372) 312(0.117) 305(0.122) C(0.0?SI ~+200(0.301} 339(0.1581 313(0-148) 306(0.10t4 A(0.0~8} ~t,00(0.164) 3t,0(0.151) 31t,(0.132) 307(0.098) 50-*(0.451} ~800[0-116)
Fig. 5. Comparison of the results of ECIA and triple antibody ELISA in the screening of sera samples from 84 Chinese pigs for trichinellosis antibodies. The tests were carried out on pooled sera samples (each based on four animals). Each test was performed in duplicate. Numbers above the figure correspond to the wells of the polystyrene plate. *indicates sample number. Numbers in brackets are the corresponding mean OD values of samples tested by triple antibody ELISA. To determine the specificity of the assays, the following heterologous, antisera were also tested: Trichuris suis (T), Metastrongylus apri (M), Gnathostoma h ispidum (G), Stephanurus dentatus ( S ), Cysticercus cellulosae (C) and Angiostrongylus cantonensis ( A ). + +, various dilutions of the positive control serum; - , negative control serum. Positive ECIA results are shown only in sample No. 338 and positive control samples (undiluted, 1:50, 1:10 and 1 : 100 dilutions).
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R.C. KO AND M.H.F. YEUNG
the OD values obtained are given in brackets beside the sample number in Fig. 5. Both assays were capable of detecting the positive sample and there were no false positive reactions. To determine the practicability of this assay as a routine screening procedure, it was applied to test serum samples from 73 pigs at an abattoir. As the pigs were from a non-endemic region, all the samples yielded negative results. However, the assay was found suitable for use in the abattoir environment, provided all the chemicals and antigen coatings were prepared beforehand. DISCUSSION
The present study shows that ECIA has a similar specificity and sensitivity as the conventional triple antibody ELISA in the detection of trichinellosis antibodies in pig serum. Parameters which affect ELISA can also influence the working of ECIA. Owing to the fact that the results of ECIA can simply be recorded on a Polaroid film, this assay will be more practical than the conventional ELISA or other colorimetric assays to be used in abattoirs, especially those with primitive conditions. With the employment of magnetic beads and all the preparative steps performed previously, ECIA can be completed as fast as 45 min. As the end point is reached very rapidly, the results can be recorded immediately after the addition of the substrate. In conventional ELISA, the colour development step usually takes about 15-30 min. Therefore, the rapidity of our system can meet the requirement of abattoirs where operation time is critical because of fast turnover rates and a substantial daily load. For example, in the abattoirs of Hong Kong, 2000-3000 pigs are commonly processed within one morning. Any immunoassay which requires more than 1 h cannot be accepted as a routine screening method. Owing to the preference of Chinese for fresh unfrozen meat, the slaughtered pigs must be delivered immediately to the retail markets. The major advantages of using magnetic beads are the dispersibility of the solid phase which can provide a relatively large surface area, fast binding rates and easy dispersal. If Dynabeads are used, the beads can be localised by magnet, thus avoiding the time consuming washing and colour development steps as in conventional ELISA. In the case of Bionectic beads, all the reagents can be prepared and dispensed into different microtitration plates beforehand. At the end of each incubation period, the beads are transferred directly from one plate into another, thus greatly reducing the processing time. The substrate reaction can also be terminated abruptly by the removal of beads. However, the great disadvantage is the current high cost of the beads. As the cost per test is dependent on the amount of antigens required, at an optimal protein: bead ratio of 150/~g per 2 ml and an antigen concentration of 0.4 /~g per well, the cost for Dynabeads is about US 30 cents per test (based on
ENZYME ASSAY TO DETECT TRICHINELLOSIS ANTIBODIES IN PIGS
109
local prices). For Bionectics beads, the cost is about US 25 cents per test. This, together with the costs for chemicals, PVC plates and instant film, will make this assay too expensive for routine large-scale screening, especially in developing countries. To improve the cost effectiveness, a cheaper substitute for the magnetic beads has to be developed. Another disadvantage is that the camera luminometer cannot provide quantification of the results as required in some research. The size and degree of whiteness of the exposed spots on the Polaroid film are directly dependent on the intensity of luminescence. But these can only be assessed visually and subjectively unless a densitometer or neutral density filters are used (Kricka and Thorpe, 1986b). However, if the Polaroid prints are copied to a black and white film, the positives will appear as dark spots on the negative. The intensity of the spots can then be quantified by a densitometer. Alternatively, luminescence can be measured by a photomultiplier or photodiode-based microplate reader. Unfortunately, the few readers which are available in the market are very expensive and are unsuitable for use in abattoirs or under field conditions. Kricka and Thorpe (1986a) used a high speed Polaroid film with 20 000 ASA (Type 612 ). However, for the present study, we used a slower T667 film with only 3000 ASA because higher speed films are not available locally. Nevertheless, both the sensitivity and contrast of the T667 film were found to be satisfactory. In fact, a slower film which is not only substantially cheaper, with its low contrast, can provide better results than T612, the contrast of which is too high to provide a graded response in image density. As the diameter of the exposed spot is dependent on the distance between the source of luminescence and the film, to achieve the m a x i m u m spot size when the shutter is opened, the bottom of the wells should be as close to the film as possible. This means that the height of the mask which houses the microplate should not be any more than a few millimetres larger than that of the plate. ACKNOWLEDGEMENT
This project was generously supported by the Croucher Foundation, Hong Kong.
REFERENCES Chan, S.W. and Ko, R.C., 1988. Comparison between standard ELISA and dot-ELISA for serodiagnosis of human trichinosis. Trans. R. Soc. Trop. Hyg., 82: 892-894. Gamble, H.R., Anderson, W.R., Graham, C.E. and Murrell; K.D., 1983. Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory-secretory antigen. Vet. Parasitol., 13:349-361.
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Gamble, H.R., Rapic, D., Marinculic, A. and Murrell, K.D., 1988. Influence cultivation conditions on specificity of excretory-secretory antigens for the immunodiagnosis of trichinellosis. Vet. Parasitol., 30: 131-137. Ko, R.C. and Yeung, M.H.F., 1989a. Specificity of ES antigens in detection of Trichinella spiralis antibodies in Chinese pigs. Trop. Biomed., 6:99-111. Ko, R.C. and Yeung, M.H.F., 1989b. Trichinella spiralis ES antigens: screening of Chinese pigs imported into Hong Kong for trichinellosis (abstract). Proceedings of the 64th Meeting of the American Society of Parasitology, Vancouver, Canada, p. 65. Kricka, L.J. and Thorpe, G.H.G., 1986a. Enhanced chemiluminescent enzyme immunoassays. Parasitol. Today, 2: 123-125. Kricka, L.J. and Thorpe, G.H.G., 1986b. Photographic detection ofchemiluminescent and bioluminescent reactions. Methods Enzymol., 133: 404-420. Schroeder, H.R., Hines, C.M., Obsborn, D.D., Moore, R.P., Hurtle, R.L., Wogoman, F.F., Rogers, R.W. and Vogelhut, P.O., 1981. Immunochemiluminometric assay for hepatitis B surface antigen. Clin. Chem., 27: 1378-1384. Thorpe, G.H.G., Haggart, R., Kricka, L.J. and Whitehead, T.P., 1984. Enhanced luminescent enzyme immunoassays for rubella antibody, immunoglobulin E and digoxin. Biochem. Biophys. Res. Commun., 119: 481-487. Thorpe, G.H.G., Kricka, L.J., Moseley, S.B. and Whitehead, T.P., 1985. Phenols as enhancers of the chemiluminescent horseradish peroxidase luminol-hydrogen perioxide reaction: application in luminescence monitored enzyme immunoassays. Clin. Chem., 31:1335-1341. Weerasekera, D.A., Kim, J.B., Barnard, G.J. and Collins, W.P., 1983. Measurement of serum thyroxine by solid-phase chemiluminescence immunoassay. Ann. Clin. Biochem., 20: 100104.
