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Magnetic bead antigen capture enzyme-linked immunoassay in microtitre trays for rapid detection of schistosomal circulating anodic antigen
Journal of Immunological Methods, 148 (1992) 1-8
I
© 1992 ElsevierScience Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06265
Magnetic bead antigen capture enzyme-linked immunoassay in microtitre trays for rapid detection of schistosomal
circulating anodic antigen S.G. G u n d e r s e n a, I. H a a g e n s e n b, T . O . J o n a s s e n b, K.J. F i g e n s e h a u b, N. de J o n g e e a n d A . M . D e e l d e r e a Department of Infectious Diseases. and b Department of Microbiology. Ullet'aal Unirersity Hospital, Oslo. Norway. and c Laboratory of Parasitology. Medical Faculty. Unirersity of Leiden. Leiden. Netherlands
(Received 10 September 1991. revisedreceived30 October 1991. accepted 17 December 1991) We have developed a new magnetic bead antigen capture enzyme-linked immunoassay for the detection of schistosomal circulating anodic antigen. The assay utilizes lgG1 monoclonal antibody coated monodisperse magnetic beads in microtitre trays fitted to a special magnet. The total test time was found to be 1-2 h, using 0.05 mg beads per well. The lower detection level was 0.7 ng A W A - T C A per ml (approximately 0.07 ng C A A per mi). Validation by sera from uninfected and Schistosoma mansoni infected Africans and Norwegians resulted in an assay specificity of 100% and sensitivity was close to 90% for cases excreting more than 100 eggs per gram faeces. At such clinically relevant levels the inter-assay CV was below 10% and photometric absorbance correlated to antigen levels was nearly linear. There was a significant correlation between the magnetic bead E I A absorbance values and the titres obtained using the previously established ELISA. The new bead assay, however, was easier znd less laborious because T C A pretreatment and the titration of positive results were unnece~ary. Key words: Magnetic bead; Microsphere; Antigen detection; ELISA; Schistosoma; Circulating anodic antigen: Tropical medicine;
Epidemiology:Monoclonalantibody, Microtiter
Introduction The need for rapid and reliable diagnosis in epidemiology and parasitic disease control has Correspondence to: S.G. Gundersen, Department of Infectiou:; Diseases, Ullevaal University Hospital, 0407 Oslo 4, Norway. Abbreriations: CAP,, circulating anodic antigen: AWATCA, trichloroaceticacid soluble fraction of adult worm antigen; TCA, trichloroacetic acid: ELISA, enzyme-linked immunosorbent assay: E1A, enzyme-linkedimmunoassay; lgGl, immunoglobulintype G-I; M, molar; PBS,phosphate-buffered saline: PEG, polyethyleneglycol;CV, coefficientof variation; mAb, monoclonal antibody; BSA, bovine serum albumin: epg, eggs per gram.
resulted in new methods for the immunological detection of parasite antigens (Voller and De Savigny, 1981; Voller, 1985). In schistosomiasis, such methods are especially needed during mass treatment campaigns at village level. Circulating anodic antigen (CAA) is one of the best studied schistosomal antigens in experimental animal models (Berggren and Weller, 1967; Gold et al., 1969; Nash et al., 1974; Deelder et al., 1976; Feidmeier et al., 1986) and in human infectious (Deelder et ai., 1980; Nash and Deelder, 1985). We have previously described an ELISA procedure for the detection of C A A which utilizes monoclonal antibodies (De Jonge et al., 1988; Deelder et al., 1989a). Using this procedure C A A
has been demonstrated in infections with different schistosome species, the serum levels of CAA correlated to worm load and circulating antigens shown to disappear after effective treatment (Decider et al., 1989a; De Jonge et al., 1989b,c, 1991). The present ELISA method is time consuming and there is a need for a simple and rapid test at village level. We have therefore applied similar principles using laagnetic beads as the solid phase. In the assays optimal kinetics are obtained whilst mobilising the beads by agitation whereas the action of a simple magnet collects the beads as a stationary solid phase during washing procedures (Nustad et al., 1988). Capture antibodies bind by strong chemical coupling to the very large exposed surface of the tosyl-activated spheres (Nustad et al., 1984, 1988). Although the use of monosized magnetic beads is well established in cellular immunology, such beads have only recently been introduced in seroimmunoassays, where they have proven to be simple, rapid and sensitive (Nustad et al., 1984, 1988; Paus and Nustad, 1989; Bormer and Nustad, 1990; Povlsen, 1991). Applications involving small sample volumes have, however, been limited. This paper reports the development of a magnetic bead antigen capture enzyme-linked immunoassay utilizing monodisperse magnetic particles (Dynabeads M280 a) and an IgGl monoclonal antibody to detect CAA in microtitre trays. The assay has been compared to the existing ELISA (Deelder et al., 1989a) using controlled antigen dilutions and sera from uninfected or Schistosoma mansoni infected Africans and Norwegians.
Materials and methods
Monoclonal antibody The production of monoclonal antibodies of the IgG1 type of the cell line 120-1B10-A against the circulating anodic antigen (CAA) of $. mansoni has been described elsewhere (Deelder et ai., 1989b). The antibody preparation was affinity purified by protein A Sepharose chromatography before use in the magnetic bead test.
Magnetic bead antigen capture EIA Tosyl-activated magnetic monodisperse particles (Dynabeads M280, Dyne,l, Oslo, Norway) were coated with anti-CAA IgGl monoclonai antibody (120-1B10-A/Leiden) as previously described (Paus and Nustad, 1989). In short, 20 mg of monoclonal antibody were incubated with l g of p-toluene sulfonyl chloride activated beads in 0.05 M borate buffer (pH 9.5) at 37°C for 20 h under rotation, resulting in a retention of 60-70% of the monoclonal antibody on the beads. Thereafter any residual binding sites on the coated beads were blocked by 2 h rotation with 2.5% (w/v) BSA in 0.1 M borate buffer (pH 9.5) and 2 h rotation in 1 M ethanolamine (pH 9.5) containing 0.1% (v/v) Tween 20 (pH 7.0), both at room temperature. The beads were finally washed with 0.1 M Tris-HCI containing 1% (w/v) BSA and 0.1% (v/v) Tween 20 (pH 7.0) and stored at 4°C in assay buffer with added 0.1% (v/v) merthiolate. In addition to investigations with various components in the assay buffers and washing solutions, investigations were performed to find the optimal temperature, reaction times and concentrations of magnetic beads and enzyme conjugated antibody. Several washing procedures and different commercial microtitre trays were also investigated. After optimization of the conditions of the test the following procedure was adopted: The assay was conducted in semi-flat bottom 96 well microtitre trays of flexible vinyl type with lids (Costar no. 6596, Badhoevedorp, Netherlands). Antigen capture and reactions with enzyme conjugated antibody were performed at 37°C while the substrate reaction occured at room temperature. All reactions were performed on a Titertek (Flow Laboratories, Irvine, UK) plateshaker in order to agitate the beads and prevent their sedimentation. Antibody-coated beads and any specific captured material were collected after reactions or during the washing steps by placing the microtitre tray on a specially adapted magnet (MPC-96, Dynal, Oslo, Norway) inside a holding frame. The supernatant was discarded after which the frame with magnet and tray was gently tapped against tissue paper to remove all fluid. The magnet was also used for manual washing of the beads by
3 moving the tray across the magnetic field and to draw the beads to the side before taking photometric absorbance readings of the substrate reactions. The washing solution contained 0.05% (v/v) Tween 20 in 50 mM Tris-HCL 150 mM NaCI, 0.5 mM MgCi 2 and 2.5 mM KCI (pH 8.0). The assay solution was the same as the washing solution with the addition of 1% (w/v) BSA. To each well were added 25/~l of a ~crum sample without any pretreatment (see under the ELISA procedure), 25/~l of assay solution and 25 ~l of a 2 mg/ml solution of beads coated with mouse anti-CAA mAb IgGl, corresponding to 0a~5 mg beads per well. After a 20 min reaction time, the tray was mounted on the magnet and supernatant removed. Without any washing 50/xl of a working solution of glycerol-solved alkaline ph0sphatase conjugated anti-CAA IgGl were added and the mixture allowed to react for a further 20 min. After removal of the supernatant the beads were washed extensively. The amount of coupled conjugate was visualised by a 30 min incubation with 50 /zl p-nitrophenyl phosphate/diethanolamine. Absorbances at 405 nm were read with a Hamilton HR 7000 spectrophotometer (Hamilton, Bonaduz, Switzerland).
Antigen capture ELISA The ELISA method was performed as described previously (Deelder et al., 1989a). In brief mierotitre trays were coated with moose ascites fluid containing lgG1 type monoclonal antibody 120-1B10-A (1/1000). The plates were incubated overnight at room temperature, washed and blocked with bovine serum albumin for 1 h at 37°C, washed again and stored at -70°C until USC.
Each serum sample was pretreated with trichloroacetic acid (De Jonge et al., 1987), centrifuged and the supernatant tested in a two-fold titration series in PBS/Tween 20/PEG. Volumes of 80/zl were added to each well, incubated for 1 h at 37°C and washed. 80 #1 of alkaline phosphatase conjugated protein A purified monocional antibody were added to each well and the plates incubated for another 1 h at 37°C. After a final washing 80 /~1 of the substrate solution p-nitrophenyl phosphate/diethanolamine were
added to each well and incubated at 37°C for 3 h, after which the absorbance was read at 405 nM with a Hamilton HR 7000 spectrophotometer.
Sen~m samples Experimental schistosomal antigen solutions. Trichloroacetic acid soluble adult worm antigen (AWA-TCA) from Leiden containing approximately 10% CAP,. (Deelder et al., 1989a) dissolved in assay buff¢r was used for optimization of the test, standard titrations and as the positive control. Norwegian blood donors. Sera were collected '~rom 32 uninfected Norwegian blood donors who had not travelled to endemic areas. Norwegia,~ imported infections. Sera were collected fron 31 patients excreting eggs of S. mansoni found by routine examinations after coming to Norway ~,om endemic countries in Africa. Faecal egg excretion was quantified by the total number of eggs found under two cover slides after formol-ether concentration (Ritchie, 1948) in two or more different stool samples. African blood donors. Sera were collected from five persons in an area of Zairc where there is no transmission of schistosomiasis (Polderman et ai., 1985). African infections. Sera were collected from 56 persons (49 Ethiopians and seven Zaireans) excreting S. mansoni eggs. Quantitative examination of egg excretion was performed on 20 mg duplicate Kato smears (Peters et al., 1980) fiom single faecal samples of the Ethiopians while 25 mg duplicates from three different faecal samples were examined from the Zairean patients. Average numbers of eggs per gram stool were calculated. The Zairean sera came from the serum bank in the Laboratory of Parasitology, Leiden, while the Ethiopian sera came from a village health programme in the Blue Nile Valley (Gundersen "et al., 1990). Sera had been stored at -20°C after separation from blood cells.
Statistics The test was validated by calculations of sensitivity, specificity and reproducibility (Vecchio, 1966; Voller and De Savigny, 1981). The interas say coefficients of variation (CV%) at different antigen levels were evaluated using the well char-
acterised AWAoTCA. The titration curves were characterised by simple linear regression equations calculated by the least squares estimates; predictions in variations were derived from the squared correlation coefficient (Pearson's r2). However, because the patient data was not normally distributed, we used non-parametric statistical methods for further evaluation. Groups of sera were compared by percentile box plots and minimum, median, 90% and maximum values. Correlations between the results of sera tested by the new magnetic bead EIA and the previously published ELISA were made by calculating Spearman's p and corresponding p values. All statistical calculations were performed with Statview software on a Macintosh S E / 3 0 personal computer.
Results
The magnetic bead E I A results were obtained as absorbances at 405 nm from which the mean absorbances of the respective negative reaction buffer blanks had been subtracted. The ELISA results were expressed as titration values. The detection levels were determined by calculating the mean of eight measurements of the negative reaction buffer plus six times the standard deviation for the magnetic bead E I A and three times the standard deviation for the ELISA. Absorbances above this level were regarded as positive in both assays at dilutions equal to or above 1 / 2 in the magnetic bead E I A and 1 / 4 in the ELISA. In the optimization experiments maximum differences between positive and negative results were found using 0.05 mg of beads per well. At higher antigen levels absorbance values after reaction times of 60 min were above those observed after 20 rain. At lower antigen levels, however, optimal reaction times seemed to have already been reached at 20 min, which was chosen both for antigen capture and conjugated antibody reactions. Similar evaluations resulted in the choice of 30 min for the substrate reaction. The standard titration curves shown in Fig. 1 demonstrated that in our hands the sensitivity of the magnetic bead EIA was similar to the ELISA,
~iO0(
c beads- EtA: Mean 121ELISA: Mean • Magnetic beads - EIA: CV%.
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~'00
AWA-TCA concentration (ng/ml)
Fig. 1. Titration curves for mean absorbances in the magnetic bead EIA compared to the EL1SA procedure at different AWA-TCAconcentrations and corresponding coefficientsof variation (CV%). Each symbol represents the mean or variation of nine consecutiveassays.
with a detection level of about 0.7 ng A W A - T C A per mi for both tests. There was a break in the curve between the five )per and the four lower magnetic bead E I A absorbances. Each of the two parts of the curve had a close to linear correlation to the A W A - T C A concentrations but there was a difference in slope and intercept for the five values above 10 ng A W A - T C A (y = 10.566x +665.264, r2=0.939), compared to the four lower values (y = 69.388x + 56.442, r 2 = 0.995). The ELISA results, however, followed a sigmoid curve (y = 14.751x + 369.029, r 2 = 0.755). The interassay CV% was lower for the magnetic bead E I A than the ELISA at most levels and below 10% at all A W A - T C A concentrations which were above 10 n g / m i . All sera from uninfected Norwegians and Zaireans were negative (Fig. 2), suggesting a specificity close to 100%. Median, 90% and maximum values of the African cases showed an increasing tendency by groups of increasing egg output (Table I). Although the sensitivity was low for those shedding less than 100 e g g s / g stool, a sensitivity close to 90% was found for more intensively infected Africans. Most Norwegian schistosomiasis cases had light infections. The three Norwegians who excreted more than 100 eggs/g stool were detected by magnetic bead EIA, while 22 of 28 low egg excretors were not detected, resulting in a very low sensitivity for this group.
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Fig. 2. Box plots displaying 10th, 25th, 50th, 75th, °A)th percentiles and outliers of the absorbances at 405 nm determined by magnetic bead EIA on samples obtained from 32 Norwegian uninfected blood donors, 31 imported Schistosomiasis mansoni cases to Norway,five African uninfected blood donors (Zaire) and 56 African Schistosomiasis mansoni cases (4° Ethiopian and seven Zairean).
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t0 too i~ MBAC-EIA: Absorbance (405 rim}x 1000
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Fig. 3. Correlation plot for the titer values of the ELISA method compared to respective absorbance values at 405 nm of the magnetic bead EIA for all the 87 sera from S. mansoni infected African and Norwegian patients shown in Fig. 2. w h o w e r e negative o r borderline in b o t h tests 26 had egg c o u n t s below 100 e g g s / g stool.
W e gave n o p r e t r e a t m e n t to sera before the magnetic b e a d E I A and did not observe any interference which could be explained by i m m u n e complexes o r nonspecific reactions o f s e r u m proteins. T h e r e was a highly significant correlation ( S p e a r m a n ' s p = 0.858, p = 0.0001) b e t w e e n abs o r b a n c e values o f magnetic b e a d E I A and E L I S A titres (Fig. 3), with trichloroacetic acid p r e t r e a t m e n t applied in the latter assay. O f the 87 infected Africans and Norwegians, 44 w e r e positive in b o t h tests, 31 w e r e negative (29) o r borderline (2) in both, while 12 w e r e positive in the magnetic bead E I A but negative in the E L I S A O f those
Discussion T h e m a g n e t i c b e a d E I A c o m b i n e s the favourable kinetic ability o f bead assays ( N u s t a d et al., 1988) with a simple enzyme-linked visualisation step. W e found a highly significant correlation b e t w e e n the results o f this new assay and the previously established E L I S A ( D e e l d e r et al., 1989a) on the same sera. W e also found an increase in m e d i a n a b s o r b a n c e values for g r o u p s with increasing egg o u t p u t , as has b e e n r e p o r t e d for the E L I S A m e t h o d ( D e J o n g e et al., 1988;
TABLE I SENSITIVITY, M1NIMUM, MEDIAN, 90% AND MAXIMUM ABSORBANCE VALUES OF THE MAGNETIC BEAD-EIA RELATED TO EGG OUTPUT GROUPS IN AFRICAN AND NORWEGIAN S. MANSON1 INFECTION Egg output group a
African S. mansoni infections b
Norwegian imported S. mansoni infections
n
n
Se %
(56) 1-100 101-400 > 400
17 22 17
70.6 90.9 88.2
Absorbance 405 nm Min.
Med.
90%
Max.
(31)
0.002 0.042 0.057
0.254 0.772 l.Ol I
0.684 1.319 2.551
0.765 1.798 2.916
28 2 1
Se %
Absorbance 405 nm Min.
Med.
90%
Max.
21.4 100 I00
0.000 0.736 -
0.043 0.759 0.332
0.501 -
1.096 0.782
a Egg output groups are constructed by quantification of the number of eggs per gram stool by the Kato method for the African and number of eggs in two coverslides after formol-ether concentration for the Norwegian patients (see text for details). h The African patients consisted of 49 Ethiopians and seven Zaireans.
Deelder et al., 1989a). In comparison, however, the magnetic bead EIA is more rapid, less laborious and involves less advanced equipment. The stability of coated beads also gives an exceptional control over the quality of the solid phase (Nustad et al., 1988). The validation characteristics of the new assay were satisfactory. Both specificity (close to 100%) and sensitivity were similar to the ELISA assay (De Jonge et al., 1988; Decider et al., 1989a), detecting AWA-TCA levels at 0.7 ng/ml (approximately 0.07 ng CAA/ml). The demonstration of antigens by the magnetic bead EIA in 12 infected persons who were ELISA negative may, in fact, indicate that the bead test is slightly more sensitive than the ELISA. Antigens were detected in 90% of persons passing more than 100 eggs per g faeces, i.e. those with clinically relevant infections (WHO, 1985). At such levels, which correspond to AWA-TCA amounts above 10 ng per mi (De Jonge et al., 1989a), the magnetic bead EIA had an acceptable interassay precision with a CV below 10%. At lower egg output levels, especially in Europeans, sensitivity seems to be equally low both for the bead assay and the ELISA (De Jonge et al., 1988; Deelder et al., 1989a). This has been explained by recent infections and the rapid clearance of immune complexes containing CAA (De Jonge et al., 1990). Moreover, established parasitological methods used in the field are unreliable for the detection of individuals with low egg output (Mott and Cline, 1980). In the present material, however, discrepancies between African and Norwegian infections might partly reflect the claim that the Ritchie method detects more light infections than the Kato method (Knight et al., 1976). Our choice of optimal conditions resulted in a present test time of 1-2 h, which compares very favourably with the 6 h or more for the ELISA procedure (Decider et al., 1989a). Early visual detection of positive substrate reactions might replace photometric endpoint absorbance readings. Under field conditions assay time would" then be less than 1 h. While the titration curve of the ELISA was sigmoid, we found an approximately linear relationship between magnetic bead EIA absorbance and antigen levels above and below 10 ng/ml. The bead assay therefore does
not appear to require any titration of positive results for more definite quantitation of antigen levels. Furthermore, the magnetic bead EIA does not demand laborious TCA pretreatment and sedimentation to remove interfering proteins and dissociate immune complexes as are necessary in the ELISA (De Jonge et al., 1987). We have not observed any such interference in the bead test. Perhaps antigen trapped in immune complexes is more accessible to the agitated beads because of their favourable thermodynamic properties, while the short reaction times may prevent nonspecific adherence of interfering proteins. The microtitre trays offer strict limitations in reaction volumes, and amounts of beads have to be optimized (Povlsen, 1991). A rather small dose of beads, 0.05 mg/well, gave maximum discrimination between negative controls and positive test sera. Larger amounts of beads can result in aggregation with antigens acting as a bridge and this was actually observed in some experiments. The small number of beads per sample is also favourable economically. Washing between antigen capture and the addition of enzyme labelled antibody was unnecessary whereas the washing before addition of the enzyme substrate was crucial. The use of automatic washers resulted in loss of beads and best washing was achieved by manual movement of the tray across the magnet. The risk of infection created by the open discharge of materials can be prevented by increasing the concentration of Tween in the reaction buffer. In individual patient care magnetic bead assays can offer a rapid diagnosis in an urgent situation (Povlsen, 1991). Schistosomiasis, however, is not usually a clinical emergency. The present assay is intended for use in epidemiological surveys undertaken in less developed countries where schistosomiasis is prevalent. For organisational reasons there is a need to obtain a rapid diagnosis during mass chemotherapy campaigns in order to individualise treatment whilst people are waiting. We conclude that the magnetic bead EIA offers a rapid and reliable assay alternative for the diagnosis of clinically relevant schistosomiasis under these conditions. An evaluation of the use of the test for such campaigns is presently being undertaken.
Acknowledgements T h i s s t u d y w a s s u p p o r t e d financially by Norw e g i a n R e s e a r c h Council for Science a n d t h e H u m a n i t i e s ( N A V F ) a n d R e s e a r c h F o r u m Ullevaal Hospital (FUS). Part o f t h e work received support from the Research and Development P r o g r a m m e " S c i e n c e a n d T e c h n o l o g y for Develo p m e n t " in t h e E u r o p e a n C o m m u n i t y . M a g n e t i c b e a d s were s u p p l i e d by Dynai, Ogle, Norway. W e t h a n k Dr. Ivar Helle, Dr. H a n s P e t t e r Torvik a n d Dr. A . M . P o l d e r m a n for t h e r e s p e c tive e x a m i n a t i o n o f N o r w e g i a n , E t h i o p i a n a n d Z a i r e a n p a t i e n t s . W e also a p p r e c i a t e v a l u a b l e advice a n d practical s u p p o r t f r o m P r o f e s s o r B]oern M y r v a n g , Mr. K]ell Skaug, Dr. Kjell N u s t a d (all Osio), Dr. L a r s A a k e Nilsson a n d Profess o r O e r j a n O u c h t e r l o n y ( b o t h G o t h e n b u r g ) . Finally we t h a n k t h e w h o l e t e a m at t h e Microbiology D e p a r t m e n t , Ullevaal Hospital, lead by Professor Kjetii Melby, for e n c o u r a g i n g collaboration.
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Mort, K.E. and Cline, B.L. (1980) Advances in epidemiology survey methodology and techniques in schistosomiasis. Bull. WHO 58, 639. Nash, T.E., Prescott, B. and Neva, F.A. (1974) The characteristics of a circulating antigen in schistosomiasis. J. lmmunol. 112, 1500. Nash, T.E. and Decider, A.M. (1985) Comparison of four schistosome excretory-secretory antigens: phenol sulfuric test active peak, cathodic circulating antigen, gut-associated proteoglycan, and circulating anodic antigen. Am. J. Trop. Med. Hyg. 34, 236. Nustad, K., Johansen, L., Ugelstad, J., Ellingsen, T. and Berge, A. (1984) Hydrophilic monodisperse particles as solid-phase material in immunoassays: Comparison of shell and core particles with compact particles. Eur. Surg. Res. 16 (suppl. 2), 80. Nustad, K., Danielsen, H., Reith, A., Funderud, S., Lea, T., Vartdal, F. and Ugelstad, J. (1988) Monodisperse polymer particles in immunoassays and cell separation. In: R.A. Tokes (Ed.), Microspheres: Medical and Biological Applications. CRC Press, Boca Raton, FL, p. 53. Paus, E. and Nustad, K. (1989) lmmunoradiometric assay for alpha gamma- and gamma gamma-enolase (neuron-specific enolase), with use of monoclonal antibodies and magnetizable polymer particles. Clin. Chem. 35, 2034.
Peters, P.A, El Alamy, M., Warren, K.S. and Mahmoud, A.A.F. (1980) Quick Kato smear for field quantitations of Schistosoma mansoni eggs. Am. J. Trop. Med. Hyg. 29, 217. Polderman, A.M., Kayiteshonga, M., Manshande, LP., Gryseels, B. and Van Sehayk, O. (1985) Historical, geological and ecological aspects of transmission of intestinal schistosmiasis in Maniema. Ann. Soe. Belg. Med. Trop. 65, 251. Povlsen, J.V. (199 i) lmmunomagnetic detection of an ti-OKT3 antibodies-an easy, fast and sensitive technique. Transplantation. 51, !118. Ritchie, L.S. (1948) An ether sedimentation technique for routine stool examination. Bull. U.S. Army Med. Dep. 8, 326. Vecchio, T.J. (1966) Predictive values of a single diagnostic test in unselected populations. New Engl. J. Meal. 274, 1171. Voller, A. (1985) Serodiagnosis of tropical parasitic diseases with special reference to 1he standardization of labelled reagent tests. Dev. Biol. Stand, 62, 3. Voller, A. and De Savigny, D. (1981) Diagnostic serology of tropical parasitic diseases. J. Immunol. Methods 46, I. WHO (1985) The control of schistosomiasis. Report of a WHO expert committee. Geneva: World Health Organization,Technical Report Series, no. 728, p. I.
I
© 1992 ElsevierScience Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06265
Magnetic bead antigen capture enzyme-linked immunoassay in microtitre trays for rapid detection of schistosomal
circulating anodic antigen S.G. G u n d e r s e n a, I. H a a g e n s e n b, T . O . J o n a s s e n b, K.J. F i g e n s e h a u b, N. de J o n g e e a n d A . M . D e e l d e r e a Department of Infectious Diseases. and b Department of Microbiology. Ullet'aal Unirersity Hospital, Oslo. Norway. and c Laboratory of Parasitology. Medical Faculty. Unirersity of Leiden. Leiden. Netherlands
(Received 10 September 1991. revisedreceived30 October 1991. accepted 17 December 1991) We have developed a new magnetic bead antigen capture enzyme-linked immunoassay for the detection of schistosomal circulating anodic antigen. The assay utilizes lgG1 monoclonal antibody coated monodisperse magnetic beads in microtitre trays fitted to a special magnet. The total test time was found to be 1-2 h, using 0.05 mg beads per well. The lower detection level was 0.7 ng A W A - T C A per ml (approximately 0.07 ng C A A per mi). Validation by sera from uninfected and Schistosoma mansoni infected Africans and Norwegians resulted in an assay specificity of 100% and sensitivity was close to 90% for cases excreting more than 100 eggs per gram faeces. At such clinically relevant levels the inter-assay CV was below 10% and photometric absorbance correlated to antigen levels was nearly linear. There was a significant correlation between the magnetic bead E I A absorbance values and the titres obtained using the previously established ELISA. The new bead assay, however, was easier znd less laborious because T C A pretreatment and the titration of positive results were unnece~ary. Key words: Magnetic bead; Microsphere; Antigen detection; ELISA; Schistosoma; Circulating anodic antigen: Tropical medicine;
Epidemiology:Monoclonalantibody, Microtiter
Introduction The need for rapid and reliable diagnosis in epidemiology and parasitic disease control has Correspondence to: S.G. Gundersen, Department of Infectiou:; Diseases, Ullevaal University Hospital, 0407 Oslo 4, Norway. Abbreriations: CAP,, circulating anodic antigen: AWATCA, trichloroaceticacid soluble fraction of adult worm antigen; TCA, trichloroacetic acid: ELISA, enzyme-linked immunosorbent assay: E1A, enzyme-linkedimmunoassay; lgGl, immunoglobulintype G-I; M, molar; PBS,phosphate-buffered saline: PEG, polyethyleneglycol;CV, coefficientof variation; mAb, monoclonal antibody; BSA, bovine serum albumin: epg, eggs per gram.
resulted in new methods for the immunological detection of parasite antigens (Voller and De Savigny, 1981; Voller, 1985). In schistosomiasis, such methods are especially needed during mass treatment campaigns at village level. Circulating anodic antigen (CAA) is one of the best studied schistosomal antigens in experimental animal models (Berggren and Weller, 1967; Gold et al., 1969; Nash et al., 1974; Deelder et al., 1976; Feidmeier et al., 1986) and in human infectious (Deelder et ai., 1980; Nash and Deelder, 1985). We have previously described an ELISA procedure for the detection of C A A which utilizes monoclonal antibodies (De Jonge et al., 1988; Deelder et al., 1989a). Using this procedure C A A
has been demonstrated in infections with different schistosome species, the serum levels of CAA correlated to worm load and circulating antigens shown to disappear after effective treatment (Decider et al., 1989a; De Jonge et al., 1989b,c, 1991). The present ELISA method is time consuming and there is a need for a simple and rapid test at village level. We have therefore applied similar principles using laagnetic beads as the solid phase. In the assays optimal kinetics are obtained whilst mobilising the beads by agitation whereas the action of a simple magnet collects the beads as a stationary solid phase during washing procedures (Nustad et al., 1988). Capture antibodies bind by strong chemical coupling to the very large exposed surface of the tosyl-activated spheres (Nustad et al., 1984, 1988). Although the use of monosized magnetic beads is well established in cellular immunology, such beads have only recently been introduced in seroimmunoassays, where they have proven to be simple, rapid and sensitive (Nustad et al., 1984, 1988; Paus and Nustad, 1989; Bormer and Nustad, 1990; Povlsen, 1991). Applications involving small sample volumes have, however, been limited. This paper reports the development of a magnetic bead antigen capture enzyme-linked immunoassay utilizing monodisperse magnetic particles (Dynabeads M280 a) and an IgGl monoclonal antibody to detect CAA in microtitre trays. The assay has been compared to the existing ELISA (Deelder et al., 1989a) using controlled antigen dilutions and sera from uninfected or Schistosoma mansoni infected Africans and Norwegians.
Materials and methods
Monoclonal antibody The production of monoclonal antibodies of the IgG1 type of the cell line 120-1B10-A against the circulating anodic antigen (CAA) of $. mansoni has been described elsewhere (Deelder et ai., 1989b). The antibody preparation was affinity purified by protein A Sepharose chromatography before use in the magnetic bead test.
Magnetic bead antigen capture EIA Tosyl-activated magnetic monodisperse particles (Dynabeads M280, Dyne,l, Oslo, Norway) were coated with anti-CAA IgGl monoclonai antibody (120-1B10-A/Leiden) as previously described (Paus and Nustad, 1989). In short, 20 mg of monoclonal antibody were incubated with l g of p-toluene sulfonyl chloride activated beads in 0.05 M borate buffer (pH 9.5) at 37°C for 20 h under rotation, resulting in a retention of 60-70% of the monoclonal antibody on the beads. Thereafter any residual binding sites on the coated beads were blocked by 2 h rotation with 2.5% (w/v) BSA in 0.1 M borate buffer (pH 9.5) and 2 h rotation in 1 M ethanolamine (pH 9.5) containing 0.1% (v/v) Tween 20 (pH 7.0), both at room temperature. The beads were finally washed with 0.1 M Tris-HCI containing 1% (w/v) BSA and 0.1% (v/v) Tween 20 (pH 7.0) and stored at 4°C in assay buffer with added 0.1% (v/v) merthiolate. In addition to investigations with various components in the assay buffers and washing solutions, investigations were performed to find the optimal temperature, reaction times and concentrations of magnetic beads and enzyme conjugated antibody. Several washing procedures and different commercial microtitre trays were also investigated. After optimization of the conditions of the test the following procedure was adopted: The assay was conducted in semi-flat bottom 96 well microtitre trays of flexible vinyl type with lids (Costar no. 6596, Badhoevedorp, Netherlands). Antigen capture and reactions with enzyme conjugated antibody were performed at 37°C while the substrate reaction occured at room temperature. All reactions were performed on a Titertek (Flow Laboratories, Irvine, UK) plateshaker in order to agitate the beads and prevent their sedimentation. Antibody-coated beads and any specific captured material were collected after reactions or during the washing steps by placing the microtitre tray on a specially adapted magnet (MPC-96, Dynal, Oslo, Norway) inside a holding frame. The supernatant was discarded after which the frame with magnet and tray was gently tapped against tissue paper to remove all fluid. The magnet was also used for manual washing of the beads by
3 moving the tray across the magnetic field and to draw the beads to the side before taking photometric absorbance readings of the substrate reactions. The washing solution contained 0.05% (v/v) Tween 20 in 50 mM Tris-HCL 150 mM NaCI, 0.5 mM MgCi 2 and 2.5 mM KCI (pH 8.0). The assay solution was the same as the washing solution with the addition of 1% (w/v) BSA. To each well were added 25/~l of a ~crum sample without any pretreatment (see under the ELISA procedure), 25/~l of assay solution and 25 ~l of a 2 mg/ml solution of beads coated with mouse anti-CAA mAb IgGl, corresponding to 0a~5 mg beads per well. After a 20 min reaction time, the tray was mounted on the magnet and supernatant removed. Without any washing 50/xl of a working solution of glycerol-solved alkaline ph0sphatase conjugated anti-CAA IgGl were added and the mixture allowed to react for a further 20 min. After removal of the supernatant the beads were washed extensively. The amount of coupled conjugate was visualised by a 30 min incubation with 50 /zl p-nitrophenyl phosphate/diethanolamine. Absorbances at 405 nm were read with a Hamilton HR 7000 spectrophotometer (Hamilton, Bonaduz, Switzerland).
Antigen capture ELISA The ELISA method was performed as described previously (Deelder et al., 1989a). In brief mierotitre trays were coated with moose ascites fluid containing lgG1 type monoclonal antibody 120-1B10-A (1/1000). The plates were incubated overnight at room temperature, washed and blocked with bovine serum albumin for 1 h at 37°C, washed again and stored at -70°C until USC.
Each serum sample was pretreated with trichloroacetic acid (De Jonge et al., 1987), centrifuged and the supernatant tested in a two-fold titration series in PBS/Tween 20/PEG. Volumes of 80/zl were added to each well, incubated for 1 h at 37°C and washed. 80 #1 of alkaline phosphatase conjugated protein A purified monocional antibody were added to each well and the plates incubated for another 1 h at 37°C. After a final washing 80 /~1 of the substrate solution p-nitrophenyl phosphate/diethanolamine were
added to each well and incubated at 37°C for 3 h, after which the absorbance was read at 405 nM with a Hamilton HR 7000 spectrophotometer.
Sen~m samples Experimental schistosomal antigen solutions. Trichloroacetic acid soluble adult worm antigen (AWA-TCA) from Leiden containing approximately 10% CAP,. (Deelder et al., 1989a) dissolved in assay buff¢r was used for optimization of the test, standard titrations and as the positive control. Norwegian blood donors. Sera were collected '~rom 32 uninfected Norwegian blood donors who had not travelled to endemic areas. Norwegia,~ imported infections. Sera were collected fron 31 patients excreting eggs of S. mansoni found by routine examinations after coming to Norway ~,om endemic countries in Africa. Faecal egg excretion was quantified by the total number of eggs found under two cover slides after formol-ether concentration (Ritchie, 1948) in two or more different stool samples. African blood donors. Sera were collected from five persons in an area of Zairc where there is no transmission of schistosomiasis (Polderman et ai., 1985). African infections. Sera were collected from 56 persons (49 Ethiopians and seven Zaireans) excreting S. mansoni eggs. Quantitative examination of egg excretion was performed on 20 mg duplicate Kato smears (Peters et al., 1980) fiom single faecal samples of the Ethiopians while 25 mg duplicates from three different faecal samples were examined from the Zairean patients. Average numbers of eggs per gram stool were calculated. The Zairean sera came from the serum bank in the Laboratory of Parasitology, Leiden, while the Ethiopian sera came from a village health programme in the Blue Nile Valley (Gundersen "et al., 1990). Sera had been stored at -20°C after separation from blood cells.
Statistics The test was validated by calculations of sensitivity, specificity and reproducibility (Vecchio, 1966; Voller and De Savigny, 1981). The interas say coefficients of variation (CV%) at different antigen levels were evaluated using the well char-
acterised AWAoTCA. The titration curves were characterised by simple linear regression equations calculated by the least squares estimates; predictions in variations were derived from the squared correlation coefficient (Pearson's r2). However, because the patient data was not normally distributed, we used non-parametric statistical methods for further evaluation. Groups of sera were compared by percentile box plots and minimum, median, 90% and maximum values. Correlations between the results of sera tested by the new magnetic bead EIA and the previously published ELISA were made by calculating Spearman's p and corresponding p values. All statistical calculations were performed with Statview software on a Macintosh S E / 3 0 personal computer.
Results
The magnetic bead E I A results were obtained as absorbances at 405 nm from which the mean absorbances of the respective negative reaction buffer blanks had been subtracted. The ELISA results were expressed as titration values. The detection levels were determined by calculating the mean of eight measurements of the negative reaction buffer plus six times the standard deviation for the magnetic bead E I A and three times the standard deviation for the ELISA. Absorbances above this level were regarded as positive in both assays at dilutions equal to or above 1 / 2 in the magnetic bead E I A and 1 / 4 in the ELISA. In the optimization experiments maximum differences between positive and negative results were found using 0.05 mg of beads per well. At higher antigen levels absorbance values after reaction times of 60 min were above those observed after 20 rain. At lower antigen levels, however, optimal reaction times seemed to have already been reached at 20 min, which was chosen both for antigen capture and conjugated antibody reactions. Similar evaluations resulted in the choice of 30 min for the substrate reaction. The standard titration curves shown in Fig. 1 demonstrated that in our hands the sensitivity of the magnetic bead EIA was similar to the ELISA,
~iO0(
c beads- EtA: Mean 121ELISA: Mean • Magnetic beads - EIA: CV%.
~ J
,t
r
TO"
"i~
~'00
AWA-TCA concentration (ng/ml)
Fig. 1. Titration curves for mean absorbances in the magnetic bead EIA compared to the EL1SA procedure at different AWA-TCAconcentrations and corresponding coefficientsof variation (CV%). Each symbol represents the mean or variation of nine consecutiveassays.
with a detection level of about 0.7 ng A W A - T C A per mi for both tests. There was a break in the curve between the five )per and the four lower magnetic bead E I A absorbances. Each of the two parts of the curve had a close to linear correlation to the A W A - T C A concentrations but there was a difference in slope and intercept for the five values above 10 ng A W A - T C A (y = 10.566x +665.264, r2=0.939), compared to the four lower values (y = 69.388x + 56.442, r 2 = 0.995). The ELISA results, however, followed a sigmoid curve (y = 14.751x + 369.029, r 2 = 0.755). The interassay CV% was lower for the magnetic bead E I A than the ELISA at most levels and below 10% at all A W A - T C A concentrations which were above 10 n g / m i . All sera from uninfected Norwegians and Zaireans were negative (Fig. 2), suggesting a specificity close to 100%. Median, 90% and maximum values of the African cases showed an increasing tendency by groups of increasing egg output (Table I). Although the sensitivity was low for those shedding less than 100 e g g s / g stool, a sensitivity close to 90% was found for more intensively infected Africans. Most Norwegian schistosomiasis cases had light infections. The three Norwegians who excreted more than 100 eggs/g stool were detected by magnetic bead EIA, while 22 of 28 low egg excretors were not detected, resulting in a very low sensitivity for this group.
Moo.
o
~_ 2500.
o
i
[
1ooo
2000¸
i
BOO
-7. • o~oo
i
IOOO
8
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.$oo
lIP
~
i
to
~
........ NOCW:M.d. (32)
Norw.$.m. (31)
Atr:bl.d. iS)
oo.
Atr.'S.m. (56)
Fig. 2. Box plots displaying 10th, 25th, 50th, 75th, °A)th percentiles and outliers of the absorbances at 405 nm determined by magnetic bead EIA on samples obtained from 32 Norwegian uninfected blood donors, 31 imported Schistosomiasis mansoni cases to Norway,five African uninfected blood donors (Zaire) and 56 African Schistosomiasis mansoni cases (4° Ethiopian and seven Zairean).
t •gogog
• ego
•
#--=.L-~
go, il"li:
g" i"g "
t0 too i~ MBAC-EIA: Absorbance (405 rim}x 1000
10000
Fig. 3. Correlation plot for the titer values of the ELISA method compared to respective absorbance values at 405 nm of the magnetic bead EIA for all the 87 sera from S. mansoni infected African and Norwegian patients shown in Fig. 2. w h o w e r e negative o r borderline in b o t h tests 26 had egg c o u n t s below 100 e g g s / g stool.
W e gave n o p r e t r e a t m e n t to sera before the magnetic b e a d E I A and did not observe any interference which could be explained by i m m u n e complexes o r nonspecific reactions o f s e r u m proteins. T h e r e was a highly significant correlation ( S p e a r m a n ' s p = 0.858, p = 0.0001) b e t w e e n abs o r b a n c e values o f magnetic b e a d E I A and E L I S A titres (Fig. 3), with trichloroacetic acid p r e t r e a t m e n t applied in the latter assay. O f the 87 infected Africans and Norwegians, 44 w e r e positive in b o t h tests, 31 w e r e negative (29) o r borderline (2) in both, while 12 w e r e positive in the magnetic bead E I A but negative in the E L I S A O f those
Discussion T h e m a g n e t i c b e a d E I A c o m b i n e s the favourable kinetic ability o f bead assays ( N u s t a d et al., 1988) with a simple enzyme-linked visualisation step. W e found a highly significant correlation b e t w e e n the results o f this new assay and the previously established E L I S A ( D e e l d e r et al., 1989a) on the same sera. W e also found an increase in m e d i a n a b s o r b a n c e values for g r o u p s with increasing egg o u t p u t , as has b e e n r e p o r t e d for the E L I S A m e t h o d ( D e J o n g e et al., 1988;
TABLE I SENSITIVITY, M1NIMUM, MEDIAN, 90% AND MAXIMUM ABSORBANCE VALUES OF THE MAGNETIC BEAD-EIA RELATED TO EGG OUTPUT GROUPS IN AFRICAN AND NORWEGIAN S. MANSON1 INFECTION Egg output group a
African S. mansoni infections b
Norwegian imported S. mansoni infections
n
n
Se %
(56) 1-100 101-400 > 400
17 22 17
70.6 90.9 88.2
Absorbance 405 nm Min.
Med.
90%
Max.
(31)
0.002 0.042 0.057
0.254 0.772 l.Ol I
0.684 1.319 2.551
0.765 1.798 2.916
28 2 1
Se %
Absorbance 405 nm Min.
Med.
90%
Max.
21.4 100 I00
0.000 0.736 -
0.043 0.759 0.332
0.501 -
1.096 0.782
a Egg output groups are constructed by quantification of the number of eggs per gram stool by the Kato method for the African and number of eggs in two coverslides after formol-ether concentration for the Norwegian patients (see text for details). h The African patients consisted of 49 Ethiopians and seven Zaireans.
Deelder et al., 1989a). In comparison, however, the magnetic bead EIA is more rapid, less laborious and involves less advanced equipment. The stability of coated beads also gives an exceptional control over the quality of the solid phase (Nustad et al., 1988). The validation characteristics of the new assay were satisfactory. Both specificity (close to 100%) and sensitivity were similar to the ELISA assay (De Jonge et al., 1988; Decider et al., 1989a), detecting AWA-TCA levels at 0.7 ng/ml (approximately 0.07 ng CAA/ml). The demonstration of antigens by the magnetic bead EIA in 12 infected persons who were ELISA negative may, in fact, indicate that the bead test is slightly more sensitive than the ELISA. Antigens were detected in 90% of persons passing more than 100 eggs per g faeces, i.e. those with clinically relevant infections (WHO, 1985). At such levels, which correspond to AWA-TCA amounts above 10 ng per mi (De Jonge et al., 1989a), the magnetic bead EIA had an acceptable interassay precision with a CV below 10%. At lower egg output levels, especially in Europeans, sensitivity seems to be equally low both for the bead assay and the ELISA (De Jonge et al., 1988; Deelder et al., 1989a). This has been explained by recent infections and the rapid clearance of immune complexes containing CAA (De Jonge et al., 1990). Moreover, established parasitological methods used in the field are unreliable for the detection of individuals with low egg output (Mott and Cline, 1980). In the present material, however, discrepancies between African and Norwegian infections might partly reflect the claim that the Ritchie method detects more light infections than the Kato method (Knight et al., 1976). Our choice of optimal conditions resulted in a present test time of 1-2 h, which compares very favourably with the 6 h or more for the ELISA procedure (Decider et al., 1989a). Early visual detection of positive substrate reactions might replace photometric endpoint absorbance readings. Under field conditions assay time would" then be less than 1 h. While the titration curve of the ELISA was sigmoid, we found an approximately linear relationship between magnetic bead EIA absorbance and antigen levels above and below 10 ng/ml. The bead assay therefore does
not appear to require any titration of positive results for more definite quantitation of antigen levels. Furthermore, the magnetic bead EIA does not demand laborious TCA pretreatment and sedimentation to remove interfering proteins and dissociate immune complexes as are necessary in the ELISA (De Jonge et al., 1987). We have not observed any such interference in the bead test. Perhaps antigen trapped in immune complexes is more accessible to the agitated beads because of their favourable thermodynamic properties, while the short reaction times may prevent nonspecific adherence of interfering proteins. The microtitre trays offer strict limitations in reaction volumes, and amounts of beads have to be optimized (Povlsen, 1991). A rather small dose of beads, 0.05 mg/well, gave maximum discrimination between negative controls and positive test sera. Larger amounts of beads can result in aggregation with antigens acting as a bridge and this was actually observed in some experiments. The small number of beads per sample is also favourable economically. Washing between antigen capture and the addition of enzyme labelled antibody was unnecessary whereas the washing before addition of the enzyme substrate was crucial. The use of automatic washers resulted in loss of beads and best washing was achieved by manual movement of the tray across the magnet. The risk of infection created by the open discharge of materials can be prevented by increasing the concentration of Tween in the reaction buffer. In individual patient care magnetic bead assays can offer a rapid diagnosis in an urgent situation (Povlsen, 1991). Schistosomiasis, however, is not usually a clinical emergency. The present assay is intended for use in epidemiological surveys undertaken in less developed countries where schistosomiasis is prevalent. For organisational reasons there is a need to obtain a rapid diagnosis during mass chemotherapy campaigns in order to individualise treatment whilst people are waiting. We conclude that the magnetic bead EIA offers a rapid and reliable assay alternative for the diagnosis of clinically relevant schistosomiasis under these conditions. An evaluation of the use of the test for such campaigns is presently being undertaken.
Acknowledgements T h i s s t u d y w a s s u p p o r t e d financially by Norw e g i a n R e s e a r c h Council for Science a n d t h e H u m a n i t i e s ( N A V F ) a n d R e s e a r c h F o r u m Ullevaal Hospital (FUS). Part o f t h e work received support from the Research and Development P r o g r a m m e " S c i e n c e a n d T e c h n o l o g y for Develo p m e n t " in t h e E u r o p e a n C o m m u n i t y . M a g n e t i c b e a d s were s u p p l i e d by Dynai, Ogle, Norway. W e t h a n k Dr. Ivar Helle, Dr. H a n s P e t t e r Torvik a n d Dr. A . M . P o l d e r m a n for t h e r e s p e c tive e x a m i n a t i o n o f N o r w e g i a n , E t h i o p i a n a n d Z a i r e a n p a t i e n t s . W e also a p p r e c i a t e v a l u a b l e advice a n d practical s u p p o r t f r o m P r o f e s s o r B]oern M y r v a n g , Mr. K]ell Skaug, Dr. Kjell N u s t a d (all Osio), Dr. L a r s A a k e Nilsson a n d Profess o r O e r j a n O u c h t e r l o n y ( b o t h G o t h e n b u r g ) . Finally we t h a n k t h e w h o l e t e a m at t h e Microbiology D e p a r t m e n t , Ullevaal Hospital, lead by Professor Kjetii Melby, for e n c o u r a g i n g collaboration.
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