$5.ao +
.00 ao4"1o1r92 © 1992 Pergamon Prea Ltd
Toxkar. Vol . 30, No. 11, pp. 1441-144e. 1992. Printed in Great Britain .
AN IDIOTYPIC-ANTI-IDIOTYPIC COMPETITIVE IMMUNOASSAY FOR QUANTITATION OF OKADAIC ACID WILLIAM S . SHFSTOWSKY, r MICHAEL A . QUILLIAM Z
and
HANNA M . SIKORSKA' *
'Raugier Bio-Tech Ltd, 8480 Blvd . St . Laurent, Montreal, Quebec, Canada H2P 2M6; and 2 National Research Council of Canada, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, Canada B3H 3Z1 (Received 30 March 1992 ; accepted 21 May 1992)
M. A. QUILLIAM and H. M . SIKoRsKA . An idiotypic-antiidiotypic competitive immunoassay for quantitation of okadaic acid. Toxicon 30, 1441-1448, 1992.-A competitive indirect enzyme-linked immunosorbent assay for the measurement of okadaic acid, a marine toxin, was developed. The assay uses a murine monoclonal anti-idiotypic antibody bearing an internal image of okadaic acid epitope to capture an anti-okadaic acid monoclonal antibody in the presence of free okadaic acid. Bound anti-okadaic acid antibody is detected with peroxidase-conjugated anti-mouse immunoglobulin antiserum. If present, free toxin will lessen the amount of anti-okadaic acid antibody binding to its corresponding anti-idiotypic antibody in a dosedependent manner that can be quantified from the standard curve. The assay permits reliable measurement of okadaic acid in the 9-81 ng/ml range. The intra- and interassay coefficients of variation in the measurement of OA in the toxin spiked mussel samples averaged 9a/o and 12a/o, respectively . The assay is rapid, accurate, reproducible and relatively simple to perform. It may be of potential use to laboratories involved in monitoring the toxin levels in plankton, seafood or sponges. W . S. SHESTOwsKY,
INTRODUCTION
measurement of okadaic acid (OA) (see Fig. 1) has recently become a topic of growing concern. Okadaic acid is a C3 , toxic polyether compound that is the major component associated with the phenomenon known as diarrhetic shellfish poisoning (DSP), and is responsible for thousands of human intoxications within the past decade caused by ingestion of shellfish contaminated with dinoflagellates such as Dinophysis sp . and Prorocentrum sp. (KUMAGAI et al ., 1986 ; MURATA et al., 1982 ; QuiLLIAM et al., 1991 ; TACHIBANA and SCHEUER, 1981 ; YASumoTo et al., 1984). There are currently several methods used for detection of okadaic acid and related compounds . The non-specific mouse or rat bioassay (YAsumoTo et al., 1985 ; KAT, 1983) is the most commonly used for monitoring contaminated shellfish. It is, however, very tedious and time-consuming to perform and imprecise in defining a causative toxin in the THE ACCURATE
" Author to whom correspondence should be addressed. 1441
1442
W . S . SHESTOWSKY et al.
R1
R2
H H Acyl
H CH3 CH3
Okadaic Acid (OA) Dinophysistoidn-1 (DTX-1) Dinophysistozin-3 (DTX-3)
MG . 1 . STxucTuxss oF oKADetc Acm, DTX-1 AND DTX-3 .
samples. The HLPC' method developed by LEE et al. (1987) is difficult to perform due to low concentrations of OA in the complex mixtures and the need to perform chemical derivatization. HPLC combined with mass spectrometry (LC-MS) has proven to be a useful method for the sensitive detection and identification of DSP toxins without the need for chemical derivatization (PLEASANCE et al., 1990); however, LC-MS instrumentation is expensive. Two radioassays have recently been described. One is an immunoassay based on the measurement of the inhibition of binding of tritium-labelled OA to polyclonal rabbit antiOA anti-serum by unlabelled OA (LEvn-E et al ., 1988). The other is a phosphatase radiobioassay that quantifies OA by specific inhibition of both protein phosphatases-1 and -2a catalytic subunits in a '~P-phosphorylase a phosphatase radioassay (HOLDS, 1991). Although this assay is very sensitive, it is difficult to perform and prone to generation of false positives by contaminants (such as NaF) that inhibit protein phosphatases. Both utilize radioactive components. A competitive ELISA, based on a monoclonal antibody developed recently by UsAGAwA et al . (1989), uses OA coupled to a protein carrier for coating of microtiter plates and peroxidase labelled anti-OA monoclonal antibody (mAb) for detection of OA. Due to the use of OA for coating, the assay is very expensive. To overcome these difficulties, two mouse monoclonal antibodies to OA, one of which is an anti-OA mAb called 6/50 (idiotype) and the other being a syngeneic anti-anti-OA mAb called 1/59 (anti-Id), have been produced . Due to the unique nature of the 1/59 antiidiotypic antibody in that it is an internal image of OA, we were able to substitute 1/59 mAb for OA and use it for coating onto solid-phase. Since 1/59 anti-Id was found to compete with OA for the same binding sites on anti-OA mAb 6/50, a competitive ELISA was developed where free OA analyte (or standard) competed with the solid-phase bound anti-idiotype for binding to a limited amount of anti-OA mAb in a liquid phase. Bound 'Abbreviatlo~anti-Id, anti4diotypic; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HPLC, high pressure liquid chromatography; HRP, horseradish peroxidase ; Id, idiotype; Ig(s), immunoglobulin(s); mAb(s), monoclonal antibody(ies); MEOH, methanol; OVAL, chicken egg ovalbumin; TS, Tris-buffered saline.
Enzyme Immunoassay for Okadaic Acid
1443
6/50 was then quantified by an enzyme-conjugated secondary antibody . The presented method provides a reliable and cost efficient means of determining the concentration of OA in marine samples. MATERIALS AND METHODS
Okadaic acid (OA) was purchased from Diagnostic Chemicals (Charlottetown, PEI, Canada). The dinophysistoxin-1 (DTX-1), provided by J. L. C. Wright (NRC Institute for Marine Biosciences), was isolated and purified from a laboratory culture of the dinollagellate species Prorocentrron lima grown at the NRC Institute for Marine Biosciences. The dinophysis-toxin-3 (DTX-3) was a generous gift from Dr T. Yasumoto (Tohoku Univ., Japan) . The anthryldiazomethane (ADAM) reagent was purchased from Molecular Probes Inc. (Eugene, Oregon). Samples of mussels contaminated with OA were kindly provided by P. Hagel (Netherlands Institute for Fishery Investigations, Ijmuiden, The Netherlands) and H. Emshohn (Ministry of Fisheries, Copenhagen, Denmark). Control mussel samples were purchased from local supermarkets. Antibody production and puryication Production of monoclonal 6/50 and 1/59 IgG, a will be described in detail elsewhere (SFE4rowsKY et al., 1991, submitted to J. Immunol.). Briefly, to produce monoclonal antibodies to OA Balb/c mice were immunized with OA-BSA conjugate, and to generate anti-idiotypic antibodies to OA Balb/c mice were injected with Bacillus Calmette-Guerin vaccine followed by several inoculations with 6/50 antibody-tuberculin purified protein derivative conjugate. In both cases, spleen cells were fused with P3X63.Ag8 .653 myeloma cells in the presence of polyethylene glycol and selected in hyposanthine, aminopterin, thymidine medium according to the standard procedure of KBrü .mt and MasrErN (1975) . IgG, antibodies were purified from ascitic fluid by affinity chromatography on Protein A-Sepharose 4B (Pharmacia, Uppsala, Sweden) essentially according to the method of Goorxa (1976) . F(ab% fragments were prepared by limited proteolysis of 1/59 IgG with pepsin (Worthington, Freehold, NJ, U.S.A ., at a pepsin to IgG ratio of 1:50 at pH 3.9. Undigested IgG and Fc fragments were removed by a passage through a Protein A-Sepharose column as described above. Preparation of shelt0h extracts Extracts were prepared according to Lm et al. (1987) as follows. Each gram of whole mussel tissue was homogenized extensively with 4 ml of aqueous 80% MEOH (Fisher, Montreal, Quebec, Canada) at room temperature using a 'Virtis' homogenizer (Virtis Co ., Gardiner, NY, U.S.A .). The homogenate was centrifuged at 3000 rpm for 15 min and the supernatant (extract) stored at -20°C until needed . For testing, extracts were diluted 1:l with distilled Hz0 to obtain a final concentration of 40% methanol. All subsequent dilutions were carried out with 40% methanol prepared in Tris-buffered saline (TS) . Spiking of mussels with OA Control mussels were spiked with OA at levels of 8 and 4 pg per gram of wet tissue to prepare Samples I and II, respectively . Both samples were then extracted with aqueous 80% methanol as described above, and then diluted for ELISA assays . HPLC analyses Extractions of mussel tissues were performed as described above. The raw extracts were then taken through the clean-up and derivatization procedure reported by LzE et al. (1987). HPLC analyses were performed on a Hewlett-Packard model 1090M instrument equipped with a ternary DR5 solvent delivery system and a HP1046A fluorescence detector . Settings for the detector were 249 nm excitation and 407 nm emission. Separations were performed at ambient temperature on a 250 x 4.6 mm ID Merck 5 ym LiChmspher 100 RP-18 column with 1 ml/min acetonitrile/water (9 :1, v/v). I"nti-ld competitive ELISA Immulon i microtiter plates (Dynatech Laboratories, Chantilly, VA, U.S.A.) were coated overnight at 4°C with F(ab')2 fragments of anti-idiotypic 1/59 IgG (5 pg/ml) in 0.05 M sodium carbonate bicarbonate buffer pH 9.6 . The unbound F(ab)2 fragments were washed off with 0.05% Tween 20/TS (v/v) and the remaining binding sites saturated with 1 % milk/TS for 1 hr at 37°C . Once the blocking agent was removed, the wells were incubated simultaneously with 50 pl of a fixed amount of 6/50 IgG (100 ng/ml) and 50A of increasing
1444
W. S. SHESTOWSKY et al.
a 0
w á é
0
. . . . . . . . . . . . .
50 100 150 200 250 6/50 Concentration (ng/mL) FIG . 2. TtrRAnoN cuRvE of 6/50 IgG AGAnasr 1/59 F(ab')2 IN ELISA. Microtiter plates coated with F(ab)= fragments of 1/59 (anti-Id) at 5,ug/ml were incubated with increasing concentations of mAb 6/50 . 6/50 binding was detected using a peroxidase-conjugated anti-mouse IgG Fc fragment specific antiserum. Each point is the mean of triplicate values. concentrations of free OA (0-1 pg/ml) in 40% MEOH for 1 hr at 37°C. After washing, a peroxidaw-conjugated anti-mouse IgG Fc fragment-specific antiserum (Sigma Chemical Co ., St Louis, MO, U.S.A .) in the 1 % milk/TS was added for 1 hr. Following removal of the unbound peroxidase conjugate by washing, the colorimetric reaction was developed upon the addition of 0.03% H=O= in 0.1% o-phenylenediamine (OPD) (Sigma), 0.1 M sodium citrate buffer, pH 7.0 . After incubation for 30 min at room temperature, the reaction was stopped by the addition of 50 A of 3 N H2SO4 (Fisher) (v/v) per well and the colour intensity measured at a wavelength of 492 nm in a Bio-Rad ELISA reader . RESULTS
Establishment of the Id-anti-Id ELISA for quantitation of OA
To determine the optimal coating concentration of 1/59, the antibody fragments coated to a plate were titrated against a fixed amount of 6/50 IgG and lowest concentration that yielded a strong and reproducible signal with a low background was selected (5 .ug/ml) . Subsequently, the 6/50 mAb was titrated against this pre-determined amount of 1/59 F(ab')2 and the concentration of 6/50 IgG contained within the linear portion of the curve (100 ng/ml) was selected (Fig. 2). To determine the antigen standard curve, OA at increasing concentrations was incubated in parallel with 6/50 IgG in microtiter wells coated with 1/59 F(ab') 2 and the bound 6/50 IgG was subsequently detected with the peroxidase-conjugated second antibody. Illustrated in Fig. 3 is a typical sigmoidal curve that exhibits linearity within 9 to 81 ng OA/ml. Assessment of the precision and validity of the Id-anti-Id ELISA
Two mussel extracts (samples I and II) were spiked with known amounts of OA as described in the Methods section and then assayed in the Id-anti-Id ELISA as described
Enzyme Immunoassay for Okadaic Acid
m
1445
0.8
e 0 0.6 a w 0 0.4 a
0 .2
OA Concentration (ng/mL) FIG. 3. OKADAIC Acm STANDARD cuRvE nN Tm Id-ANTI-Id ELISA. Microtiter wells coated with mAb 1/59 F(ab)2 (5 pg/ml) were incubated with increasing concentrations of OA and a fixed concentration of mAb 6/50 (100 ng/ml) . Bound antibody was detected as described under Fig. 1. Each point is the mean of triplicate values and the error bars show the 95% confidence intervals.
TARIE 1. REPRODUCIBILITY AND ACCURACY OF THE ELISA FOR SAMPLES CONTAINING OICADAIC ACID'
Samplest Intra-assay variability
Interassay variability
Day of assaying
Tissue Number of concentration$ assays (Jug/9)
CV§ (%)
I
2 3 4 5
3 3 3 2
8.16±0.64 8.40±1 .30 9.20±0.98 7.20±1 .00
8 16 10 14
11
2 3 4 5
3 3 3 2
4.72±0.11 3.50±0.14 4.76±0.20 3.30±0.42
2 4 5 13
I
1-5
5
8.48±0.79
9
II
1-5
5
4.04±0 .62
15
Five assays were done on successive days. To evaluate intra-assay variation, three assays were done on days 2, 3 and 4; two assays were done on day 5. In all assays, determinations were carried out in triplicate . Interassay variation was measured by comparing the mean of each sample on the five successive days . tSamples were methanol extracts of fresh mussels prepared according to IxE el al. (1987) . Samples I and II were spiked with pure okadaic acid as described in the Methods section at the 8 pg/g (1) and 4 pg/g (II) levels . t Mean ±S.D . S.D . x 100. § Coefficient of variation = mean
1446
W . S . SHESTOWSKY et al.
á
25 50 76 100 OA Concentration (na/mL) Fxt . 4 . VALtDrry of ELISA. Comparison of Id-anti-Id ELISA with HPLC method for quantitation of OA in mussel samples. The mussel extracts, prepared in 80% methanol as described in the Methods section, were assayed by the HPLC method (h et al., 1987) and then diluted for ELISA measurements to yield the values plotted as the abscissa. Individual ELISA measurements are shown. (--) First-order linear regression (r2 = 0 .9439); (-) ideal result .
above. The intra-assay coefficient of variation (CV) was determined by testing each sample in duplicate in at least two assays/day on four independent occasions. The values thus obtained ranged between 8 and 16% for sample I, and 2 and 13% for sample II. The interassay CV% was evaluated by assaying the two samples on five successive days. The interassay CV% ranged from 9 to 15% (Table 1). Several mussel tissue samples, currently being evaluated as possible reference materials for the analysis of okadaic acid, were extracted and analyzed by HPLC using the method of LEE et al. (1987). The concentrated extracts were diluted and tested in ELISA. Figure 4 shows a good correlation between results of the two methods.
Specificity of the Id-anti-Id ELISA
The specificity of the Id-anti-Id ELISA was examined against two toxins closely related to OA, dinophysistoxin-1 (DTX-1) and dinophysistoxin-3 (DTX-3) (see Fig. 1). The ELISA results demonstrated in Fig. 5 indicate that anti-OA mAb 6/50 recognizes DTX-1 but not DTX-3. 6/50 IgG shows 8% to 16% cross-reactivity with DTX-1 . Thus, provided that a sample is known to contain only DTX-1 (e.g. plankton), this toxin could be quantified in the range of 25-200 ng/ml in the Id-anti-Id ELISA for OA using a DTX-1 standard curve. The assay would not be accurate, however, for quantitation of either OA or DTX-1 in mixed samples (containing both toxins), since the 6/50 antibody cannot accurately distinguish OA from DTX-1 . For shellfish contaminated with several DSPs the Id-anti-Id ELISA is semi-quantitative in that it will detect either of the toxins at concentrations higher than 25 ng/g wet tissue.
Enzyme Immunoassay for Okadaic Acid
1447
o OA DTX-t v DTX-9
Toxin Concentration (ng/mQ Fia . 5 . Specmcrnr op Id-Arrn-ID ELISA. Microtitration wells coated with F(ab)2 fragments of 1/59 IgG were incubated with increasing amounts of either OA (-m-), DTX-1 (-1-), or DTX-3 (-A-) in the presence of a constant amount (100 ng/ml) of 6/50 IgG as described in the Materials and Methods section. Bound 6/50 IgG was detected with peroiddase-anti-mouse IgG Fc fragment specific antiserum and expressed as percent bound according to the formula: A,. nm in the presence of toxin-background A . nm A. 2 nm in the absence of toxin-background A Z nm
DISCUSSION
Using two mouse monoclonal antibodies described here, optimal conditions for a competitive ELISA for determination of OA content in contaminated marine samples have been established. The assay, performed using conventional ELISA methodology, demonstrates the sensitivity and reliability required for the accurate quantitation of OA levels in marine extracts . The unique feature of the assay described is that OA used to coat microtiter plates was replaced with an anti-idiotypic mAb bearing the internal image of OA (Ab2ß) . This mAb mimics the action of OA both in an ELISA and in a phosphatase bioassay, and thus can serve as a surrogate of OA (SII1NTowsKY et al., manuscript in preparation) . The main advantage of using an internal image mAb in place of OA is that murine monoclonal antibodies can be produced in large quantities at a low cost while OA is prohibitively expensive. Secondly, due to OA's small size (mol. wt approximately 800) direct adsorption of OA to a solid-phase may either alter or destroy certain epitopes on the molecule so chemical coupling of OA to a protein carrier is necessary (Dmtxs et al., 1986). This may also have deleterious effects on its antigenicity . Anti-idiotypic antibody, however, can be coated to the solid phase directly without loss of immunogenecity . In comparison with the immunoassays described in the Introduction, the Id-anti-Id ELISA is much simpler, faster (3 hr) and cheaper to perform ($2.34 vs. $25.00 US/assay) since it consumes very small amounts of OA (standards only). Although slightly less
1448
W. S. SHESTOWSKY et al.
sensitive (9-81 ng/ml) than these radioassays, it is still able to detect OA levels far below quarantine levels (200 ng/g) set by the Japanese governmental agencies . The assay will also detect DTX-1, but semi-quantitatively and only when present at concentrations higher than 25 ng/ml. To quantify DTX-1, an OA standard curve should be replaced by a DTX-1 standard curve, but only when it is known that no OA is present in the sample since 6/50 antibody has 10-fold higher affinity for OA than for DTX-1 . Acknowledgements-The authors would like to thank the National Research Council of Canada for its financial support under the Contribution Agreement No. CA-949-9-0008. The technical assistance of W. R. HARDsTAFF is gratefully acknowledged . Special acknowledgements are addressed to Dr TAKEStn YAsumoTO from Tohoku University, Japan, and Dr JEFFREY L. C. WRIGHT from NRC Institute for Marine Biosciences, Halifax, Canada, for their generous gifts of DTX-3 and DTX-1, and to Ms ANGELA ARSENEAU and Ms LINDA SHERIDAN for their excellent editorial assistance in the preparation of this manuscript . REFERENCES DmRtL4, S. E., BUTLER, J. E. and Rwrn3esoN, H. B. (1986) Altered recognition of surface-adsorbed compared to antigen-bound antibodies in the ELISA. Mot. Immunol. 23, 403411 . GGDiNG, J. W. (1976) Conjugation of antibodies with fluorochromes : modification to the standard methods. J. Immunol. Meth . 13,215--226 . Hof u s, C. F. B. (1991) Liquid chromatography-linked protein phosphatase bioassay; a highly sensitive marine bioscreen for okadaic acid and related diarrhetic shellfish toxins. Toxicon 29, 469477 . KAT, M. (1983) Diarrhetic mussel poisoning in the Netherlands related to the dinoflagellate Dinophysis acuminata. Antonie van leeuwenhoek 49, 417427 . KBrD.Et, B. and MILSTEIN, C. (1975) Continuous culture of fused cells secreting antibody of predefined specificity . Nature 256, 495497 . KUMAGAI, M., YANAGi, T., MURATA, M., YASUMOTO, T., KAT, M., LAssus, P. and RODRjQuEz-VAzQuEz, J. A. (1986) Okadaic acid as the causative toxin of diarrhetic shellfish poisoning in Europe. Agric. Biol. Chem . 50, 2853-2857 . LEE, J. S., YANAor, T., KENMA, R. and YAsumaTo, T. (1987) Fluorometric determination of diarrhetic shellfish toxins by high-performance liquid chromatography. Agric. Biol . Chem . 51, 877-881. LEwNE, L., Futur, H., YAMADA, K., OnKA, M., GmcA, H. and VAN VUNAKrs, H. (1988) Production of antibodies and development of a radioimmunoassay for okadaic acid . Toxicon 26, 1123-1128. MURATA, M., SmuATANi, M., SUGITANi, H., OsmmA, Y. and YASumoTo T. (1982) Isolation and structural elucidation of the causative toxin of the diarrhetic shellfish poisoning. Bull. Jpn. Soc. Sci. Fish . 49, 549-552. PLEASANCE, S., QUILT.TAM, M.A ., DEFRErrAs, A. S. W., MARK, J. C. and CEMHEr.LA, A. D. (1990) Ion-spray mass spectrometry of marine toxins II. Analysis of diarrhetic shellfish toxins in plankton by liquid chromatography/ mass spectrometry. Rapid Commmr. Mass Spectrometry 4, 206-213. QuiLmAM, M. A., GILGAN, M. W., PLEASANCE, S., DEFRmAs, A. S. W., DOUGLAS, D., FRrrz, L., Hu, T., MARK, J. C., SMYTH, C. and WRIGHT, J. L. C. (1991) Confirmation of an incident of diarrhetic shellfish poisoning in Eastern Canada. Presented at the Fifth International Conference on Toxic Marine Phytoplankton, Newport, Rhode Island, 28 October-l November 1991 (in press). TACmRANA, K. and SCHEUER, P. J. (1981) Okadaic acid, a cytotoxic polyether from two marline sponges of the genus Halichondria . J. Am. Chem. Soc. 103, 2469-2470 . USAoAwA, T., NnmmuRA, M., ITOH, Y., UDA, T. and YASUMoTo, T. (1989) Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai. Toxicon 27, 1323-1330. YASumoTo, T., MURATA, M., OSHIMA, Y., MATSUMOTO, G. L. and CLARDY, J. (1984) Diarrhetic shellfish toxin poisoning. In : Seafood Toxins ACS Symp . Ser No . 262, pp . 207-214 (RAGELrs, E. P., Ed.) . Washington, DC : American Chemical Society. YAsumoTo, T., MURATA, M., OstmtA, M., SANG, M., MATSUMOTO, G. K. and CLARDY, J. (1985) Diarrhetic shellfish toxins . Tetrahedron 41, 1019-1025.
.00 ao4"1o1r92 © 1992 Pergamon Prea Ltd
Toxkar. Vol . 30, No. 11, pp. 1441-144e. 1992. Printed in Great Britain .
AN IDIOTYPIC-ANTI-IDIOTYPIC COMPETITIVE IMMUNOASSAY FOR QUANTITATION OF OKADAIC ACID WILLIAM S . SHFSTOWSKY, r MICHAEL A . QUILLIAM Z
and
HANNA M . SIKORSKA' *
'Raugier Bio-Tech Ltd, 8480 Blvd . St . Laurent, Montreal, Quebec, Canada H2P 2M6; and 2 National Research Council of Canada, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, Canada B3H 3Z1 (Received 30 March 1992 ; accepted 21 May 1992)
M. A. QUILLIAM and H. M . SIKoRsKA . An idiotypic-antiidiotypic competitive immunoassay for quantitation of okadaic acid. Toxicon 30, 1441-1448, 1992.-A competitive indirect enzyme-linked immunosorbent assay for the measurement of okadaic acid, a marine toxin, was developed. The assay uses a murine monoclonal anti-idiotypic antibody bearing an internal image of okadaic acid epitope to capture an anti-okadaic acid monoclonal antibody in the presence of free okadaic acid. Bound anti-okadaic acid antibody is detected with peroxidase-conjugated anti-mouse immunoglobulin antiserum. If present, free toxin will lessen the amount of anti-okadaic acid antibody binding to its corresponding anti-idiotypic antibody in a dosedependent manner that can be quantified from the standard curve. The assay permits reliable measurement of okadaic acid in the 9-81 ng/ml range. The intra- and interassay coefficients of variation in the measurement of OA in the toxin spiked mussel samples averaged 9a/o and 12a/o, respectively . The assay is rapid, accurate, reproducible and relatively simple to perform. It may be of potential use to laboratories involved in monitoring the toxin levels in plankton, seafood or sponges. W . S. SHESTOwsKY,
INTRODUCTION
measurement of okadaic acid (OA) (see Fig. 1) has recently become a topic of growing concern. Okadaic acid is a C3 , toxic polyether compound that is the major component associated with the phenomenon known as diarrhetic shellfish poisoning (DSP), and is responsible for thousands of human intoxications within the past decade caused by ingestion of shellfish contaminated with dinoflagellates such as Dinophysis sp . and Prorocentrum sp. (KUMAGAI et al ., 1986 ; MURATA et al., 1982 ; QuiLLIAM et al., 1991 ; TACHIBANA and SCHEUER, 1981 ; YASumoTo et al., 1984). There are currently several methods used for detection of okadaic acid and related compounds . The non-specific mouse or rat bioassay (YAsumoTo et al., 1985 ; KAT, 1983) is the most commonly used for monitoring contaminated shellfish. It is, however, very tedious and time-consuming to perform and imprecise in defining a causative toxin in the THE ACCURATE
" Author to whom correspondence should be addressed. 1441
1442
W . S . SHESTOWSKY et al.
R1
R2
H H Acyl
H CH3 CH3
Okadaic Acid (OA) Dinophysistoidn-1 (DTX-1) Dinophysistozin-3 (DTX-3)
MG . 1 . STxucTuxss oF oKADetc Acm, DTX-1 AND DTX-3 .
samples. The HLPC' method developed by LEE et al. (1987) is difficult to perform due to low concentrations of OA in the complex mixtures and the need to perform chemical derivatization. HPLC combined with mass spectrometry (LC-MS) has proven to be a useful method for the sensitive detection and identification of DSP toxins without the need for chemical derivatization (PLEASANCE et al., 1990); however, LC-MS instrumentation is expensive. Two radioassays have recently been described. One is an immunoassay based on the measurement of the inhibition of binding of tritium-labelled OA to polyclonal rabbit antiOA anti-serum by unlabelled OA (LEvn-E et al ., 1988). The other is a phosphatase radiobioassay that quantifies OA by specific inhibition of both protein phosphatases-1 and -2a catalytic subunits in a '~P-phosphorylase a phosphatase radioassay (HOLDS, 1991). Although this assay is very sensitive, it is difficult to perform and prone to generation of false positives by contaminants (such as NaF) that inhibit protein phosphatases. Both utilize radioactive components. A competitive ELISA, based on a monoclonal antibody developed recently by UsAGAwA et al . (1989), uses OA coupled to a protein carrier for coating of microtiter plates and peroxidase labelled anti-OA monoclonal antibody (mAb) for detection of OA. Due to the use of OA for coating, the assay is very expensive. To overcome these difficulties, two mouse monoclonal antibodies to OA, one of which is an anti-OA mAb called 6/50 (idiotype) and the other being a syngeneic anti-anti-OA mAb called 1/59 (anti-Id), have been produced . Due to the unique nature of the 1/59 antiidiotypic antibody in that it is an internal image of OA, we were able to substitute 1/59 mAb for OA and use it for coating onto solid-phase. Since 1/59 anti-Id was found to compete with OA for the same binding sites on anti-OA mAb 6/50, a competitive ELISA was developed where free OA analyte (or standard) competed with the solid-phase bound anti-idiotype for binding to a limited amount of anti-OA mAb in a liquid phase. Bound 'Abbreviatlo~anti-Id, anti4diotypic; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HPLC, high pressure liquid chromatography; HRP, horseradish peroxidase ; Id, idiotype; Ig(s), immunoglobulin(s); mAb(s), monoclonal antibody(ies); MEOH, methanol; OVAL, chicken egg ovalbumin; TS, Tris-buffered saline.
Enzyme Immunoassay for Okadaic Acid
1443
6/50 was then quantified by an enzyme-conjugated secondary antibody . The presented method provides a reliable and cost efficient means of determining the concentration of OA in marine samples. MATERIALS AND METHODS
Okadaic acid (OA) was purchased from Diagnostic Chemicals (Charlottetown, PEI, Canada). The dinophysistoxin-1 (DTX-1), provided by J. L. C. Wright (NRC Institute for Marine Biosciences), was isolated and purified from a laboratory culture of the dinollagellate species Prorocentrron lima grown at the NRC Institute for Marine Biosciences. The dinophysis-toxin-3 (DTX-3) was a generous gift from Dr T. Yasumoto (Tohoku Univ., Japan) . The anthryldiazomethane (ADAM) reagent was purchased from Molecular Probes Inc. (Eugene, Oregon). Samples of mussels contaminated with OA were kindly provided by P. Hagel (Netherlands Institute for Fishery Investigations, Ijmuiden, The Netherlands) and H. Emshohn (Ministry of Fisheries, Copenhagen, Denmark). Control mussel samples were purchased from local supermarkets. Antibody production and puryication Production of monoclonal 6/50 and 1/59 IgG, a will be described in detail elsewhere (SFE4rowsKY et al., 1991, submitted to J. Immunol.). Briefly, to produce monoclonal antibodies to OA Balb/c mice were immunized with OA-BSA conjugate, and to generate anti-idiotypic antibodies to OA Balb/c mice were injected with Bacillus Calmette-Guerin vaccine followed by several inoculations with 6/50 antibody-tuberculin purified protein derivative conjugate. In both cases, spleen cells were fused with P3X63.Ag8 .653 myeloma cells in the presence of polyethylene glycol and selected in hyposanthine, aminopterin, thymidine medium according to the standard procedure of KBrü .mt and MasrErN (1975) . IgG, antibodies were purified from ascitic fluid by affinity chromatography on Protein A-Sepharose 4B (Pharmacia, Uppsala, Sweden) essentially according to the method of Goorxa (1976) . F(ab% fragments were prepared by limited proteolysis of 1/59 IgG with pepsin (Worthington, Freehold, NJ, U.S.A ., at a pepsin to IgG ratio of 1:50 at pH 3.9. Undigested IgG and Fc fragments were removed by a passage through a Protein A-Sepharose column as described above. Preparation of shelt0h extracts Extracts were prepared according to Lm et al. (1987) as follows. Each gram of whole mussel tissue was homogenized extensively with 4 ml of aqueous 80% MEOH (Fisher, Montreal, Quebec, Canada) at room temperature using a 'Virtis' homogenizer (Virtis Co ., Gardiner, NY, U.S.A .). The homogenate was centrifuged at 3000 rpm for 15 min and the supernatant (extract) stored at -20°C until needed . For testing, extracts were diluted 1:l with distilled Hz0 to obtain a final concentration of 40% methanol. All subsequent dilutions were carried out with 40% methanol prepared in Tris-buffered saline (TS) . Spiking of mussels with OA Control mussels were spiked with OA at levels of 8 and 4 pg per gram of wet tissue to prepare Samples I and II, respectively . Both samples were then extracted with aqueous 80% methanol as described above, and then diluted for ELISA assays . HPLC analyses Extractions of mussel tissues were performed as described above. The raw extracts were then taken through the clean-up and derivatization procedure reported by LzE et al. (1987). HPLC analyses were performed on a Hewlett-Packard model 1090M instrument equipped with a ternary DR5 solvent delivery system and a HP1046A fluorescence detector . Settings for the detector were 249 nm excitation and 407 nm emission. Separations were performed at ambient temperature on a 250 x 4.6 mm ID Merck 5 ym LiChmspher 100 RP-18 column with 1 ml/min acetonitrile/water (9 :1, v/v). I"nti-ld competitive ELISA Immulon i microtiter plates (Dynatech Laboratories, Chantilly, VA, U.S.A.) were coated overnight at 4°C with F(ab')2 fragments of anti-idiotypic 1/59 IgG (5 pg/ml) in 0.05 M sodium carbonate bicarbonate buffer pH 9.6 . The unbound F(ab)2 fragments were washed off with 0.05% Tween 20/TS (v/v) and the remaining binding sites saturated with 1 % milk/TS for 1 hr at 37°C . Once the blocking agent was removed, the wells were incubated simultaneously with 50 pl of a fixed amount of 6/50 IgG (100 ng/ml) and 50A of increasing
1444
W. S. SHESTOWSKY et al.
a 0
w á é
0
. . . . . . . . . . . . .
50 100 150 200 250 6/50 Concentration (ng/mL) FIG . 2. TtrRAnoN cuRvE of 6/50 IgG AGAnasr 1/59 F(ab')2 IN ELISA. Microtiter plates coated with F(ab)= fragments of 1/59 (anti-Id) at 5,ug/ml were incubated with increasing concentations of mAb 6/50 . 6/50 binding was detected using a peroxidase-conjugated anti-mouse IgG Fc fragment specific antiserum. Each point is the mean of triplicate values. concentrations of free OA (0-1 pg/ml) in 40% MEOH for 1 hr at 37°C. After washing, a peroxidaw-conjugated anti-mouse IgG Fc fragment-specific antiserum (Sigma Chemical Co ., St Louis, MO, U.S.A .) in the 1 % milk/TS was added for 1 hr. Following removal of the unbound peroxidase conjugate by washing, the colorimetric reaction was developed upon the addition of 0.03% H=O= in 0.1% o-phenylenediamine (OPD) (Sigma), 0.1 M sodium citrate buffer, pH 7.0 . After incubation for 30 min at room temperature, the reaction was stopped by the addition of 50 A of 3 N H2SO4 (Fisher) (v/v) per well and the colour intensity measured at a wavelength of 492 nm in a Bio-Rad ELISA reader . RESULTS
Establishment of the Id-anti-Id ELISA for quantitation of OA
To determine the optimal coating concentration of 1/59, the antibody fragments coated to a plate were titrated against a fixed amount of 6/50 IgG and lowest concentration that yielded a strong and reproducible signal with a low background was selected (5 .ug/ml) . Subsequently, the 6/50 mAb was titrated against this pre-determined amount of 1/59 F(ab')2 and the concentration of 6/50 IgG contained within the linear portion of the curve (100 ng/ml) was selected (Fig. 2). To determine the antigen standard curve, OA at increasing concentrations was incubated in parallel with 6/50 IgG in microtiter wells coated with 1/59 F(ab') 2 and the bound 6/50 IgG was subsequently detected with the peroxidase-conjugated second antibody. Illustrated in Fig. 3 is a typical sigmoidal curve that exhibits linearity within 9 to 81 ng OA/ml. Assessment of the precision and validity of the Id-anti-Id ELISA
Two mussel extracts (samples I and II) were spiked with known amounts of OA as described in the Methods section and then assayed in the Id-anti-Id ELISA as described
Enzyme Immunoassay for Okadaic Acid
m
1445
0.8
e 0 0.6 a w 0 0.4 a
0 .2
OA Concentration (ng/mL) FIG. 3. OKADAIC Acm STANDARD cuRvE nN Tm Id-ANTI-Id ELISA. Microtiter wells coated with mAb 1/59 F(ab)2 (5 pg/ml) were incubated with increasing concentrations of OA and a fixed concentration of mAb 6/50 (100 ng/ml) . Bound antibody was detected as described under Fig. 1. Each point is the mean of triplicate values and the error bars show the 95% confidence intervals.
TARIE 1. REPRODUCIBILITY AND ACCURACY OF THE ELISA FOR SAMPLES CONTAINING OICADAIC ACID'
Samplest Intra-assay variability
Interassay variability
Day of assaying
Tissue Number of concentration$ assays (Jug/9)
CV§ (%)
I
2 3 4 5
3 3 3 2
8.16±0.64 8.40±1 .30 9.20±0.98 7.20±1 .00
8 16 10 14
11
2 3 4 5
3 3 3 2
4.72±0.11 3.50±0.14 4.76±0.20 3.30±0.42
2 4 5 13
I
1-5
5
8.48±0.79
9
II
1-5
5
4.04±0 .62
15
Five assays were done on successive days. To evaluate intra-assay variation, three assays were done on days 2, 3 and 4; two assays were done on day 5. In all assays, determinations were carried out in triplicate . Interassay variation was measured by comparing the mean of each sample on the five successive days . tSamples were methanol extracts of fresh mussels prepared according to IxE el al. (1987) . Samples I and II were spiked with pure okadaic acid as described in the Methods section at the 8 pg/g (1) and 4 pg/g (II) levels . t Mean ±S.D . S.D . x 100. § Coefficient of variation = mean
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W . S . SHESTOWSKY et al.
á
25 50 76 100 OA Concentration (na/mL) Fxt . 4 . VALtDrry of ELISA. Comparison of Id-anti-Id ELISA with HPLC method for quantitation of OA in mussel samples. The mussel extracts, prepared in 80% methanol as described in the Methods section, were assayed by the HPLC method (h et al., 1987) and then diluted for ELISA measurements to yield the values plotted as the abscissa. Individual ELISA measurements are shown. (--) First-order linear regression (r2 = 0 .9439); (-) ideal result .
above. The intra-assay coefficient of variation (CV) was determined by testing each sample in duplicate in at least two assays/day on four independent occasions. The values thus obtained ranged between 8 and 16% for sample I, and 2 and 13% for sample II. The interassay CV% was evaluated by assaying the two samples on five successive days. The interassay CV% ranged from 9 to 15% (Table 1). Several mussel tissue samples, currently being evaluated as possible reference materials for the analysis of okadaic acid, were extracted and analyzed by HPLC using the method of LEE et al. (1987). The concentrated extracts were diluted and tested in ELISA. Figure 4 shows a good correlation between results of the two methods.
Specificity of the Id-anti-Id ELISA
The specificity of the Id-anti-Id ELISA was examined against two toxins closely related to OA, dinophysistoxin-1 (DTX-1) and dinophysistoxin-3 (DTX-3) (see Fig. 1). The ELISA results demonstrated in Fig. 5 indicate that anti-OA mAb 6/50 recognizes DTX-1 but not DTX-3. 6/50 IgG shows 8% to 16% cross-reactivity with DTX-1 . Thus, provided that a sample is known to contain only DTX-1 (e.g. plankton), this toxin could be quantified in the range of 25-200 ng/ml in the Id-anti-Id ELISA for OA using a DTX-1 standard curve. The assay would not be accurate, however, for quantitation of either OA or DTX-1 in mixed samples (containing both toxins), since the 6/50 antibody cannot accurately distinguish OA from DTX-1 . For shellfish contaminated with several DSPs the Id-anti-Id ELISA is semi-quantitative in that it will detect either of the toxins at concentrations higher than 25 ng/g wet tissue.
Enzyme Immunoassay for Okadaic Acid
1447
o OA DTX-t v DTX-9
Toxin Concentration (ng/mQ Fia . 5 . Specmcrnr op Id-Arrn-ID ELISA. Microtitration wells coated with F(ab)2 fragments of 1/59 IgG were incubated with increasing amounts of either OA (-m-), DTX-1 (-1-), or DTX-3 (-A-) in the presence of a constant amount (100 ng/ml) of 6/50 IgG as described in the Materials and Methods section. Bound 6/50 IgG was detected with peroiddase-anti-mouse IgG Fc fragment specific antiserum and expressed as percent bound according to the formula: A,. nm in the presence of toxin-background A . nm A. 2 nm in the absence of toxin-background A Z nm
DISCUSSION
Using two mouse monoclonal antibodies described here, optimal conditions for a competitive ELISA for determination of OA content in contaminated marine samples have been established. The assay, performed using conventional ELISA methodology, demonstrates the sensitivity and reliability required for the accurate quantitation of OA levels in marine extracts . The unique feature of the assay described is that OA used to coat microtiter plates was replaced with an anti-idiotypic mAb bearing the internal image of OA (Ab2ß) . This mAb mimics the action of OA both in an ELISA and in a phosphatase bioassay, and thus can serve as a surrogate of OA (SII1NTowsKY et al., manuscript in preparation) . The main advantage of using an internal image mAb in place of OA is that murine monoclonal antibodies can be produced in large quantities at a low cost while OA is prohibitively expensive. Secondly, due to OA's small size (mol. wt approximately 800) direct adsorption of OA to a solid-phase may either alter or destroy certain epitopes on the molecule so chemical coupling of OA to a protein carrier is necessary (Dmtxs et al., 1986). This may also have deleterious effects on its antigenicity . Anti-idiotypic antibody, however, can be coated to the solid phase directly without loss of immunogenecity . In comparison with the immunoassays described in the Introduction, the Id-anti-Id ELISA is much simpler, faster (3 hr) and cheaper to perform ($2.34 vs. $25.00 US/assay) since it consumes very small amounts of OA (standards only). Although slightly less
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W. S. SHESTOWSKY et al.
sensitive (9-81 ng/ml) than these radioassays, it is still able to detect OA levels far below quarantine levels (200 ng/g) set by the Japanese governmental agencies . The assay will also detect DTX-1, but semi-quantitatively and only when present at concentrations higher than 25 ng/ml. To quantify DTX-1, an OA standard curve should be replaced by a DTX-1 standard curve, but only when it is known that no OA is present in the sample since 6/50 antibody has 10-fold higher affinity for OA than for DTX-1 . Acknowledgements-The authors would like to thank the National Research Council of Canada for its financial support under the Contribution Agreement No. CA-949-9-0008. The technical assistance of W. R. HARDsTAFF is gratefully acknowledged . Special acknowledgements are addressed to Dr TAKEStn YAsumoTO from Tohoku University, Japan, and Dr JEFFREY L. C. WRIGHT from NRC Institute for Marine Biosciences, Halifax, Canada, for their generous gifts of DTX-3 and DTX-1, and to Ms ANGELA ARSENEAU and Ms LINDA SHERIDAN for their excellent editorial assistance in the preparation of this manuscript . REFERENCES DmRtL4, S. E., BUTLER, J. E. and Rwrn3esoN, H. B. (1986) Altered recognition of surface-adsorbed compared to antigen-bound antibodies in the ELISA. Mot. Immunol. 23, 403411 . GGDiNG, J. W. (1976) Conjugation of antibodies with fluorochromes : modification to the standard methods. J. Immunol. Meth . 13,215--226 . Hof u s, C. F. B. (1991) Liquid chromatography-linked protein phosphatase bioassay; a highly sensitive marine bioscreen for okadaic acid and related diarrhetic shellfish toxins. Toxicon 29, 469477 . KAT, M. (1983) Diarrhetic mussel poisoning in the Netherlands related to the dinoflagellate Dinophysis acuminata. Antonie van leeuwenhoek 49, 417427 . KBrD.Et, B. and MILSTEIN, C. (1975) Continuous culture of fused cells secreting antibody of predefined specificity . Nature 256, 495497 . KUMAGAI, M., YANAGi, T., MURATA, M., YASUMOTO, T., KAT, M., LAssus, P. and RODRjQuEz-VAzQuEz, J. A. (1986) Okadaic acid as the causative toxin of diarrhetic shellfish poisoning in Europe. Agric. Biol. Chem . 50, 2853-2857 . LEE, J. S., YANAor, T., KENMA, R. and YAsumaTo, T. (1987) Fluorometric determination of diarrhetic shellfish toxins by high-performance liquid chromatography. Agric. Biol . Chem . 51, 877-881. LEwNE, L., Futur, H., YAMADA, K., OnKA, M., GmcA, H. and VAN VUNAKrs, H. (1988) Production of antibodies and development of a radioimmunoassay for okadaic acid . Toxicon 26, 1123-1128. MURATA, M., SmuATANi, M., SUGITANi, H., OsmmA, Y. and YASumoTo T. (1982) Isolation and structural elucidation of the causative toxin of the diarrhetic shellfish poisoning. Bull. Jpn. Soc. Sci. Fish . 49, 549-552. PLEASANCE, S., QUILT.TAM, M.A ., DEFRErrAs, A. S. W., MARK, J. C. and CEMHEr.LA, A. D. (1990) Ion-spray mass spectrometry of marine toxins II. Analysis of diarrhetic shellfish toxins in plankton by liquid chromatography/ mass spectrometry. Rapid Commmr. Mass Spectrometry 4, 206-213. QuiLmAM, M. A., GILGAN, M. W., PLEASANCE, S., DEFRmAs, A. S. W., DOUGLAS, D., FRrrz, L., Hu, T., MARK, J. C., SMYTH, C. and WRIGHT, J. L. C. (1991) Confirmation of an incident of diarrhetic shellfish poisoning in Eastern Canada. Presented at the Fifth International Conference on Toxic Marine Phytoplankton, Newport, Rhode Island, 28 October-l November 1991 (in press). TACmRANA, K. and SCHEUER, P. J. (1981) Okadaic acid, a cytotoxic polyether from two marline sponges of the genus Halichondria . J. Am. Chem. Soc. 103, 2469-2470 . USAoAwA, T., NnmmuRA, M., ITOH, Y., UDA, T. and YASUMoTo, T. (1989) Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai. Toxicon 27, 1323-1330. YASumoTo, T., MURATA, M., OSHIMA, Y., MATSUMOTO, G. L. and CLARDY, J. (1984) Diarrhetic shellfish toxin poisoning. In : Seafood Toxins ACS Symp . Ser No . 262, pp . 207-214 (RAGELrs, E. P., Ed.) . Washington, DC : American Chemical Society. YAsumoTo, T., MURATA, M., OstmtA, M., SANG, M., MATSUMOTO, G. K. and CLARDY, J. (1985) Diarrhetic shellfish toxins . Tetrahedron 41, 1019-1025.
