of LFI for PSP toxin detection, however, presented some unique problems. Antibodies must recognize about twenty analogues of saxitoxin (STX) in a variety of.
254 MIST AlertTM: A RAPID ASSAY FOR PARALYTIC
SHELLFISH
POISONING
TOXINS
Maurice V. Laycock’, Joanne F. Jellett’, Elizabeth R. Belland’, Pamela C. Bishop’, Brigitte L. Thériault’, Audra L. Russell-Tattrie’, Michael A. Quilliam*, Allan D. Cembella*, Robert C. Richards ’ Jellett Biotek Limited, P.O. Box 790, Dartmouth, Nova Scotia, CANADA, B2Y 327; * National Research Council of Canada, Institute for Marine Biosciences, 1411 Oxford St., Halifax, Nova Scotia, CANADA, B3H 321 ABSTRACT Jellett Biotek Ltd. has developed a rapid fïeld test kit to screen for paralytic shellfish poisoning (PSP) toxins. The new test, called MIST AlertTM, provides a qualitative (positive/negative) indication of the presence of PSP toxins in less than 20 min. It is designed as a screening method for regulatory labs to eliminate negative samples, thereby leaving a smaller number of positive samples to be tested with more sophisticated and time-consuming quantitative methods. Due to its simplicity and speed, MIST AlertTM cari also be used in other applications such as shellfish harvest management and toxin research. The test is based on easy-to-use lateral flow immunochromatographic (LFI) test strips. The sensitivity of the test to several analogues of saxitoxin (STX) and neosaxitoxin (NEO) was investigated. The toxin analogues STX, GTX2/3 and Cl/2 epimeric mixtures, Bl and decarbamoyl saxitoxin (dc-STX) were detectable at concentrations of approximately 200 nM. The test was somewhat less sensitive to the NI-hydroxy derivatives NE0 and GTX1/4, requiring concentrations of 400 and 600 nM, respectively, to give a substantially positive test. Al1 toxins were detectable within or close to the regulatory limit of 80 pg saxitoxin dihydrochloride equivalents per 100 g of tissue (= 1075 nM STX) using the AOAC extraction procedure.
100 g of tissue, may be encountered, rather than a simple presence or absence of toxic analyte. An ideal assay should indicate a positive result at or above the regulatory limit of 80 ug STX(2HCl)eq per 100 g of tissue and a clear negative below this value. TO develop and test a new method for detecting PSP toxins an adequate supply of the purified toxins was essential, especially one involving antibodies where toxin-protein conjugates were required for both immunizations and to form capture lines on the test strips. Furthermore, the production of LFI test strips with reliable and uniform responses required specialized equipment to spray membranes with conjugated antibodies and toxins. Component concentrations were adjusted to give an average response to different analogues for each batch of several thousand test strips. Different capture line conjugates and mixtures of antibodies raised against different analogues give different responses SOthat test strips cari be designed to suit toxin profiles found in different regions. In this publication we describe the response of a batch of LFI test strips to purified PSP toxins to illustrate some characteristics of this product.
C
Harmful Algal Blooms 2000 Hallegraeff, G.M., Blackbum, S.I., Bolch, C.J. and Lewis, R.J. (eds) IntergovemmentalOceanographicCommissionof UNESCO 2001
S
10
INTRODUCTION Variations of the intraperitoneal mouse bioassay, now standardized by the AOAC [l] procedure, have been used to detect paralytic toxins in shellfish for more than sixty years. Some alternative assays have been developed but none, until now, were well suited for fïeld use. Antibody methods offered the best approach for the development of an easy-to-use, reliable and inexpensive test kit for PSP toxin detection. One approach to implementing an immunoassay is by lateral flow immuno-chromatography (LFI). In such an assay, all the components are incorporated into a test strip, SOthat it is only necessary to add a sample extract to initiate the sequence of reactions. As a result, LFI tests require no special expertise or laboratory equipment in their use. Consequently, this technology has found many applications, such as in home pregnancy test kits. The use of LFI for PSP toxin detection, however, presented some unique problems. Antibodies must recognize about twenty analogues of saxitoxin (STX) in a variety of complex matrices extracted from different shellfish tissues and species. Furthermore, in naturally contaminated shellfish a wide range of concentrations, from 0 to >105 ug saxitoxin equivalents (STXeq) per
T
Absorption Pad
Membrane
r
I
Sampleand ConiuaatePad
1 80 pg of toxin per 100g
E
I t’
T
R
No Toxin I Fig. 1. Diagram of the MIST AlertTM test strip. The components are aligned with two cassettes containing developed test strips. Strips consist of an absorption pad, a membrane striped with a mixture of toxin analogues (the T line on the right) and an antibody detection reagent (the C line on the left) a sample pad and a conjugate pad containing the antibodies. The visible T line indicates absence of toxin in the sample and no line indicates presence of toxin. The C line indicates that the sample fluid has sufficiently resuspended and mobilized the antibody colour complex.
255
3200
Toxin Concentrations in nM 1600 800 400 200 100 50
RESULTS 25
Fig. 2. Dilution series for neosaxitoxin. Eight test strips were attached to a tard and sample pads dipped into microtitre plate Wells containing 100 uL of running buffer with different concentrations of toxin.
MATERIALS
AND METHODS
PSP toxins were purifïed from toxic strains of Aiexandrium by methods described previously [2]. Toxin concentrations were measured by capillary electrophoresis [3] and calibrated with certified calibration solutions (National Research Council of Canada). Complete test strips were mounted in plastic cassettes (see Fig. 1) or glued on cards for dilution series experiments. Sample extracts were diluted to 1/5 with Jellett Biotek’s commercial running buffer solution. Test strips mounted on cards were spaced SOthat the sample pads fitted into microtitre plate Wells. The fïrst well in a row contained 200 PL of buffer solution with 3200 nM toxin. The other seven Wells across the plate contained 100 pL of buffer. Serial dilutions were made by transferring and mixing 100 pL from each well into the next giving a dilution series from 3200 to 25 nM. TO represent the results graphically each tard of strips was scanned and intensities of test lines were integrated using a BioRad Model-GS-690.
Sensitivities
When a solution is added to the sample pad of a test strip, antibody and coloured particles are carried along the membrane and, in the absence of free toxin, bind to the toxin conjugate at the T line. Any free toxin present in the sample solution blocks antigen-binding sites SOthat the antibodies cannot bind to the conjugated toxin at the T line. If the toxin concentration is high enough, the T line is not visible. There is no competition of free toxin at the C line which Will always appear, providing antibodies and coloured particles have properly bound and moved forward. Non-specific interference is indicated by the absence of a C line and cari be caused by interfering compounds in shellfish extracts or factors such as salt content or pH effects. A visible C line shows that the test performed properly. Therefore, decrease in intensity of the T line compared to a negative control indicates the presence of PSP toxins in the sample. The concentration at which this occurs is different for each toxin. In Fig. 3 it cari be seen that STX eliminated the T line at concentrations greater than 200 nM, whereas for NE0 and GTX 1/4 (equimolar epimeric mixture) concentrations in excess of 400 and 600 nM, respectively, were necessary to eliminate the T line. Due to interference from compounds in extracts of shellfish tissues it was necessary to dilute them to at least 1/5 with the running buffer. This also allowed for pH and salt concentrations for optimal test performance. The samples cari consist of shelltïsh extracts, phytoplankton extracts or solutions of pure toxins diluted in the running buffer. An aliquot of 100 uL is applied to each test strip and the sample liquid resuspends the antibodies and colour complex located in the sample and conjugate pads, whereupon liquid migrates along the membrane through the capture lines and into the absorbant pad. In the presence of free PSP toxin a weak T line often forms but it gradually disappears by competition between free and bound toxin.
of MIST AlertTM to PSP Toxins
400
Toxin Concentration
600
nM
Fig. 3. Plots of test line intensities of purified PSP toxins from dilution series experiments.
256 DISCUSSION Mist AiertTM is designed primarily to provide a rapid test for screening shellfish extracts to reduce the number of more accurate, quantitative analyses, most of which are negative. MIST AlertT”, therefore, should not be used to replace analytical methods. Although the cost of mice is relatively low, testing laboratories have to charge according to the overall costs of an analytical laboratory. Concerns over the use of whole mammal bioassays as subjects for seafood testing are increasing and restricting the use of mice in routine screening programs for shellfish toxins. Other methods for PSP toxin analysis, such as liquid chromatography, are even more expensive than the mouse bioassay, particularly for small numbers of samples. The MIST AlertTM test strips cari be made relatively inexpensively in thousands using equipment designed for the production of LFI test strips. Production in large batches also ensures their reproducibility. For routine testing each test strip is mounted in a cassette (Fig. 1) and about 100 pL of extract diluted with a buffer solution is added to the sample well. This simple operation cari be made without laboratory equipment and an eye-dropper cari be used. Alter twenty minutes, persistence of a strong T line indicates that any PSP toxins in the sample are at concentrations less than the regulatory limit. Toxin concentrations at or above the equivalent of 80 pg STX(2HCI) per 100 g tissue (1075 nM for STX) in an AOAC extract eliminate the toxin line and further testing should be done on those samples. The response of the test to different PSP toxins is unequal, due to specificities of the antibodies and competition for the bound toxin on the T line. Experiments with T lines of mixed toxin conjugates showed that this approach to detecting the entire family of PSP toxins was not as effective in eliminating the line as using a blend of antibodies. Cross-reactivities of our antibodies were generally similar to those reported by other groups [eg. 4, 51 in that antibodies against NE0 recognized NE0 and its analogues and those raised against STX recognized STX and its analogues, with good recognition of the respective GTX derivatives within the two groups. Fig. 3 shows that sensitivities of the test strip to STX, GTX2/3, dcSTX, C1/2, and Bl were around 200 nM, allowing for a five fold dilution to obtain test concentrations of 1075 nM, equivalent to an AOAC extract of shellfïsh tissue at the regulatory limit of 80 pg STXeq per 100 g. Responses to NE0 and GTX1/4 were less than for STX analogues, indicating that a higher proportion of anti-NE0 antibodies is needed for optimal response. The slope of the intensity-concentration curve for GTX1/4 in Fig. 3 shows that the intensity of the T line gradually decreased with increasing toxin concentration. In fact, for a11 of the analogues that were tested, the slopes increased with higher sensitivity. The non-Nlhydroxy derivatives STX, GTX2/3 and dcSTX yielded the sharpest distinction between a line and no line, whereas as shown in Fig. 2, the T line persisted over a wider range in response to different concentrations of NEO. Interpretation of a single test result is difficult
where a line remains visible, even though it may be faint in comparison to that of a control test. Such ambiguities occur near the detection limit of the strip assay for those analogues, but usually not at concentrations signifïcantly above the regulatory limit. Sensitivities of the assay to the N-sulfo-carbamoyl epimers Cl and C2, and Bl (GTXS) were intermediate between those for STX and NEO. This result is different from that previously reported for immunoassays in which cross-reactivities were very low to sulfamate toxins [4, 5,]. Because sulfate is easily lost from the N-21 position of sulfamate toxins, tare was taken to ensure there were no detectable amounts of carbamate toxins present. Analysis by capillary electrophoresis showed no detectable contamination with STX or GTX2/3; if present, these components were well below the detection limits of the strip assay. Although toxicities of sulfamate toxins are relatively low compared to the carbamate group, they are easily converted to more toxic carbamate analogues. Therefore, the ability to detect sulfamate toxins is an advantage, especially in a screening test. Test strips are currently being produced using the antibody mixture yielding cross-reactivities shown here for purified toxins. The composition of the solutions that are provided with the kit for dilution have been developed for different applications and are propriatary. Because of the differing affmities of the antibody mixture for the individual analogues it is not possible to provide a definitive detection limit for the mixed toxin profiles in shellfish tissues. Depending on the averuge toxin profile in any geographic area, the detection limit of the test units produced with the current antibody mixture Will be in the range of 100-800 nM which corresponds to 7-60 pg STXeq per 100 g tissue. Jellett Biotek Will endeavour to produce an antibody mixture with more uniform cross-reactivities among the analogues for future versions. MIST AlertTM is currently undergoing field trials in several locations, including Alaska and Great Britain, with comparisons to HPLC data and mouse bioassays. The results of this survey Will be the subject of a future publication. REFERENCES of Officia1 Analytical Chemists, 1 . Association Procedure 49.9.01, in: Officia1 Methods of Analysis, 16th ed., P. Cunniff, ed. (AOAC International, Arlington), (1995) P.Thibault, S.W.Ayer, and J.A. 2. M.V.Laycock, Walter. Natural Toxins 2, 175-l 83 (1994) S.Pleasance, and M.V.Laycock, J. 3. P.Thibault, Chromatog. 542,483-501 (1991) 4. A.D.Cembella, Y.Parent, D.Jones, G.Lamoureux, in: Toxic Marine Phytoplankton, E.Granéli, B. Sundstrom, L.Edler, D.M.Anderson, eds. (Elsevier, New York), pp. 339-344 (1989) 5. X.Huang, K-H.Hsu, and F.S.Chu, J.Agric.Food Chem. 44, 1029-1035 (1996)
