Development of a quantitative ELISA for screening fodder for corynetoxins
A report for the Rural Industries Research and Development Corporation
By Dr Khin Than, Dr Steve Colegate and Dr John Edgar
October 2002 RIRDC Publication No 02/118 RIRDC Project No. CSA-3A
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© 2002 Rural Industries Research and Development Corporation. All rights reserved.
ISBN 0642 58517 2 ISSN 1440-6845
Development of a quantitative ELISA for screening fodder for corynetoxins Publication No. 02/118 Project No. CSA-3A
The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report.
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Researcher Contact Details Dr Khin Than Plant Toxins Research Group CSIRO Livestock Industries Australian Animal Health Laboratory Private Bag 24, Geelong, Vic 3220
Phone: (03) 5227 5731 Fax: (03) 5227 5555 Email:
[email protected]
In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.
RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Website:
02 6272 4539 02 6272 5877
[email protected]. http://www.rirdc.gov.au
Published in October 2002 Printed on environmentally friendly paper by Canprint
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Foreword The aim of this project was to develop an enzyme-linked-immuno-sorbent-assay (ELISA) to screen fodder for the presence of corynetoxins. The assay will help ensure that Australian fodder does not cause poisoning of livestock that could lead to long-term disruptions to trade. Australian fodder contaminated by corynetoxins, the natural chemicals that cause annual ryegrass toxicity (ARGT), poisoned beef and dairy cattle in Japan in 1996. Japanese authorities questioned the safety of meat and milk from livestock exposed to corynetoxins and they banned Australian fodder until a quality assurance (QA) program was established. The QA protocol adopted by the industry is based on ELISA detection of the bacterial source of the corynetoxins, Rathayibacter (Clavibacter) toxicus. Whilst it is assumed that the presence or absence of the bacterium is a good indication of the presence of corynetoxins, this may not necessarily be the case. It needs to be conclusively demonstrated that a low bacterial antigen ELISA result does indeed relate to a low corynetoxin level. Furthermore, because the bacterium does not produce corynetoxins continuously throughout its life cycle, there is a high chance of rejecting fodder based on the bacterium antigen ELISA result, whereas the level of corynetoxins may in fact be very low and thereby the fodder would be safe for stock. Since the corynetoxins, and not the bacterium per se, cause ARGT, an ELISA for routinely measuring corynetoxins in fodder will provide greater assurance that further damaging incidents of corynetoxin poisoning do not jeopardise export and domestic markets for fodder. If further incidents of ARGT occur, a long-term ban on exports of fodder is expected and reactivated concerns with food safety could flow on to affect markets for Australian meat, dairy products and grain. The corynetoxin ELISA, developed during the two-year period of this project, was shown to be effective for application to fodder samples. It offers a means of determining the safety of fodder for stock. The corynetoxin ELISA will now need to undergo further testing in several laboratories using the same extraction procedure and common extracts for both bacterial (R. toxicus) and corynetoxin ELISAs to ensure its usefulness to the Fodder Industry. Inter-laboratory testing should be conducted to ensure that the corynetoxin ELISA meets acceptable standards for QA of fodder. This project was funded from industry revenue which is matched by funds provided by the Federal Government. The present report, a new addition to RIRDC’s diverse range of over 800 research publications, forms part of our Fodder Crops R&D program, which aims to facilitate the development of a sustainable and profitable Australian fodder industry. Most of our publications are available for viewing, downloading or purchasing online through our website: downloads at www.rirdc.gov.au/reports/Index.htm purchases at www.rirdc.gov.au/eshop
Simon Hearn Managing Director Rural Industries Research and Development Corporation
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Acknowledgements The researchers would like to thank Veronica Olsen, Neil Anderton, Peter Cockrum and Yu Cao for their technical and scientific expertise critical to the successful completion of this project. The researchers would also like to thank Michael Mackie and Rosa Thijse from Gilmac (WA) Pty Ltd, Malcolm May and Andrew Parkinson from Balco Australia Pty Ltd (SA), and Kym Shardlow from Golden Plains Fodder Australia Pty Ltd (SA) for supplying the fodder samples used in the development and application of the ELISA.
Abbreviations annual ryegrass toxicity (ARGT) bovine serum albumin (BSA) electrospray ionisation mass spectrometry (esiMS) enzyme-linked-immuno-sorbent-assay (ELISA) evaporative light scattering detector (ELSD) high performance liquid chromatography (HPLC) mass spectrometry (MS) photo diode array detector (PDAD) quality assurance (QA) South Australian Research and Development Institute (SARDI)
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Contents Foreword ............................................................................................................................................... iii Acknowledgements............................................................................................................................... iv Abbreviations........................................................................................................................................ iv Contents.................................................................................................................................................. v Executive Summary ............................................................................................................................. vi Introduction ........................................................................................................................................... 1 Objectives ............................................................................................................................................... 2 Methodology .......................................................................................................................................... 2 Isolation and characterisation of pure corynetoxins............................................................................ 2 Generation of anti-corynetoxin antisera .............................................................................................. 3 Format and conditions for corynetoxin ELISA ................................................................................... 3 Extraction of corynetoxins from fodder .............................................................................................. 6 Validation of the indirect, competitive corynetoxin ELISA ............................................................... 7 Results .................................................................................................................................................... 7 HPLC and indirect, competitive .......................................................................................................... 7 Corynetoxin levels in fodder samples ................................................................................................. 7 Technology transfer ............................................................................................................................ 13 Recommendation ................................................................................................................................. 13 Implications.......................................................................................................................................... 13 Appendix I............................................................................................................................................ 14 Corynetoxin ELISA........................................................................................................................... 14 References ............................................................................................................................................ 24
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Executive Summary Objectives The aim of the project was to develop an ELISA for screening fodder for corynetoxins that will be useful in quality assurance of fodder and help to ensure that corynetoxin-contaminated fodder does not cause annual ryegrass toxicity (ARGT).
Background Hay contaminated with corynetoxins causes ARGT in livestock, a problem found only in Australia. Oaten hay from Australia was responsible for several incidents of ARGT in Japan that led to export restrictions. Current QA of hay uses an ELISA for the bacterial source of the corynetoxins rather than the toxins themselves. Since the corynetoxins, and not the bacterium per se, cause ARGT, the bacterium antigen ELISA is an indirect way of ensuring that fodder is safe. Indeed, some hay that shows a strong response when assessed by the bacterial assay may be safe for stock. It was therefore considered desirable to establish a method for detecting and measuring the corynetoxins in order to improve management of the problem. Patented CSIRO technology associated with anti-corynetoxin antibodies was considered to be suitable for the purpose, but needed to be adapted for specific application to fodder.
Research Antisera against the corynetoxins can be induced in sheep when they are injected with a modified form of the corynetoxins conjugated to a protein. Existing supplies of anti-corynetoxin antisera and newly generated sera were assessed for their suitability in a corynetoxin ELISA for application to fodder samples. The conditions for conducting the ELISA were optimised. This involved research into coating microtitre plates with modified toxins, selection of assay buffers and assay conditions. The potential for extractives from the fodder to interfere with the assay was also assessed. The solvent for extracting trace levels of toxin present was selected with regard to cost, safety and efficiency. The ELISA was then validated against a confirmatory HPLC assay before it was applied to 113 fodder samples.
Outcomes The assay was shown to be effective for application to fodder samples. It was found to be capable of accurately measuring corynetoxins down to 40 parts per billion in fodder. It offers a means of determining the safety of fodder for stock. The corynetoxin ELISA will now need to undergo further testing in several laboratories to ensure its usefulness to the Fodder Industry. An officer from Agriculture WA has been trained in its use. Similar training will be offered to SARDI and Agri-Food Technology, Werribee, Victoria.
Recommendation Inter-laboratory testing of the corynetoxin ELISA should be conducted by CSIRO, Agriculture WA, SARDI and Agrifood Technology using the same extraction protocol and common fodder extracts for both bacterial and corynetoxin ELISAs to ensure that the corynetoxin ELISA meets acceptable standards for QA of fodder. Preliminary assessment, jointly done with Agriculture WA, has shown that the aqueous cyclodextrin solutions, used for extraction of corynetoxins from fodder, were also suitable for use with the R. toxicus ELISA. This will allow both corynetoxin and R. toxicus ELISAs to be used to assess the same extracts and will facilitate comparison of these two ELISAs as indicators of the safety of fodder for livestock.
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Implications After inter-laboratory testing, during which some fine tuning is anticipated, has demonstrated the robustness and suitability of the ELISA for routine application to fodder samples, it will be available to the Fodder Industry for use in assuring the safety of fodder with the potential to cause ARGT. It is expected to enable the Fodder Industry to better manage the problem of ARGT and to protect the industry from future trade disruptions. The reagents and standards needed for the assay are specialised products and their future availability needs to be established if the assay proves to be important for the Fodder Industry.
Publications Than KA, Cao Y, Michalewicz A, Cockrum PA, Olsen V and Edgar JA (1999). Novel quantitative ELISA for corynetoxins. Society for Food and Agricultural Immunology’s 5th International Conference: Agri-Food Antibodies ’99; Sep 14 - Sep 17, 1999: Norwich, U.K. [Abstr.].
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Introduction Australian oaten hay, contaminated by corynetoxins, the chemicals causing ARGT, poisoned beef and dairy cattle in Japan in 1996. This caused Japanese authorities to stop imports of Australian fodder until a quality assurance program was established to prevent more poisoning incidents from occurring. The Australian Fodder Industry has established a protocol that is aimed at reducing the likelihood of another poisoning incident. Any further corynetoxin poisoning incident in Japan, caused by Australian fodder, is expected to result in a long-term loss of markets for Australian fodder. The QA protocol currently in place is based on detection of the bacterium Rathayibacter (Clavibacter) toxicus in fodder. R. toxicus is a plant pathogenic bacterium that colonises certain grasses, including annual ryegrass, and it is the source of the corynetoxins. The bacterium is readily detectable on infected annual ryegrass in August/September but the production of corynetoxins does not reach significant and poisonous levels until late October or early November as the grass dries. Therefore, fodder cut early, before corynetoxin production begins, may be positive for the presence of R. toxicus but may be non-toxic to livestock. This means that some non-poisonous fodder may be rejected if the R. toxicus assay alone is used for QA. Since the corynetoxins are the cause of ARGT, it is also necessary to prove beyond reasonable doubt that low bacterial antigen ELISA results do in fact represent low levels of corynetoxins all the time. Therefore, for any QA protocol, an ELISA based on measuring corynetoxins is, at least, a necessary complementary assay to the existing ELISA based on measuring R. toxicus. During the 1996 incidents of poisoning caused by Australian oaten hay, the Japanese authorities were particularly concerned about the safety of milk and meat from livestock consuming corynetoxincontaminated fodder. The risk to consumers of such products is not known and will require many years of intensive research to establish. In the mean time there is reason for consumers to be concerned about the safety of products from animals that have consumed corynetoxin-contaminated fodder. If long term, low level exposure of humans to corynetoxins in animal products is shown to cause health problems, then an auditable record of the level of exposure of the animals to corynetoxins via fodder would assist the Industry. CSIRO has been the principal research provider in regard to the isolation, detection and quantitation of corynetoxins. It has developed, and maintains a capacity to detect and measure these extremely poisonous natural chemicals using techniques such as high performance liquid chromatography (HPLC) and mass spectrometry (MS). These methods, while valuable as unequivocal confirmatory assays for corynetoxins, are totally inappropriate for screening bulk commodities, such as fodder, for corynetoxins. At present, these methods are time-consuming and require costly equipment and highly skilled technicians. The sensitivity of these methods is also inadequate for QA of fodder, except in cases of gross corynetoxin contamination. During research to develop a vaccine to protect livestock from corynetoxin poisoning (ARGT), CSIRO developed methods for generating antibodies against the corynetoxins, demonstrated the use of several different anti-corynetoxin antisera to detect trace level of corynetoxins and patented the details of the inventions involved. These discoveries provide the basis for generating rapid screening assays for corynetoxins that may be applicable to the various commodities and products subjected to corynetoxin contamination, including fodder.
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Objectives To demonstrate an enzyme-linked-immuno-sorbent-assay (ELISA) for screening fodder for corynetoxins that will be useful in quality assurance of fodder and help to ensure that corynetoxin-contaminated fodder does not cause annual ryegrass toxicity (ARGT)
Methodology Isolation and characterisation of pure corynetoxins Pure corynetoxins are required as authentic standards to confirm specificity and sensitivity of the ELISA and to calibrate each ELISA plate, using known dilutions of the authentic corynetoxins, to allow for plateto-plate variations and as an indication of reliability of the assay results.
Methods employed Source of corynetoxins: Screenings of wheat and barley obtained in Western Australia and transported to Victoria in sealed drums in a sealed container, according to quarantine requirements, have been used as a source of corynetoxins. Manual screening of this material, under appropriate occupational health and safety work conditions, provided a bacterial gall-rich concentrate that was shown to be extremely poisonous to sheep. Analysis by HPLC indicated that it contained 83 micrograms of corynetoxins per gram. Eleven oral doses of 170gms/day/sheep of this material, given over 6 days in the first week and 5 days in the second week, killed 9 of 10 unvaccinated sheep. Extraction of corynetoxins: The gall concentrate was extracted several times with dichloromethane to remove lipophilic, non-corynetoxin material. It was then extracted with water to remove water soluble, noncorynetoxin material. Finally the corynetoxins were extracted by steeping the screenings several times with aqueous methanol. The aqueous methanol corynetoxin-containing extract was concentrated by evaporation under vacuum. Isolation of corynetoxins: Using small aliquots of the above concentrate, a number of previously used and several new procedures have been examined to further purify the corynetoxins. High performance liquid chromatography and the experimental ELISAs were used to monitor the effect of these procedures on corynetoxin concentration. For example, mild alkaline hydrolysis was employed to destroy and remove other ampipathic co-extractives such as triglycerides while acid precipitation of corynetoxins also contributed to concentrating the corynetoxins and removing non-corynetoxin material from the initial concentrate. Of various other methods examined, solid phase adsorption and elution using custom-made reversed-phase C18 silica was also found to be a useful purification step. Further exploratory purification has involved the use of normal-phase silica columns. More than sixty milligrams of corynetoxins were extracted and purified. Characterisation and quantitation were done using high performance liquid chromatography/photo diode array detector/electrospray ionisation mass spectrometry (HPLC/PDAD/esiMS) or by high performance liquid chromatography /evaporative light scattering detector (HPLC/ELSD). The suitability of the corynetoxins sample for use in the ELISA development and as an authentic standard has been confirmed using the two corynetoxin ELISAs (Figures 1 and 2) described below.
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Generation of anti-corynetoxin antisera Twenty five sheep were purchased and divided into 6 groups. Each group was injected with a different tunicamycin derivative-protein conjugate. Tunicamycins are closely related to the corynetoxins in chemical structure and are toxicologically equivalent. Each conjugate was administered as an emulsion with 5 mg DEAE-dextran + 0.5 mg Quil A saponin/dose in Montanide 888 oil. The total volume of 2 ml was injected under the skin at the back of the neck. The initial injections were followed by regular booster injections. Ten ml of blood from each sheep was collected before and after each injection to monitor the antibody response. The antibody suite produced by each animal is unique and thus, only antisera from some of the animals were expected to be suitable for developing an assay for detecting and measuring corynetoxins in fodder. It was therefore necessary to inject a number of animals to find one or two with the right antibodies. Nine sheep previously vaccinated for a previous research at CSIRO were also reboosted regularly. The suitability of the anti-corynetoxin antibodies produced by each sheep for corynetoxin ELISA development was determined by screening antisera for corynetoxin binding. When the sheep produced strong antibody responses to corynetoxins, 300ml of blood were collected for further analysis. CSIRO has screened, ranked and selected several promising antisera, that can be diluted more than x 4000 and still show an ELISA optical density difference of 70-80% between 0 and 1.6 nanograms corynetoxins or tunicamycins /100µl/well of ELISA plate.
Format and conditions for corynetoxin ELISA Quantitation of toxins The purity of the corynetoxins, recently produced by CSIRO, was estimated by HPLC. Individual corynetoxins U17a and H17a were isolated by preparative HPLC. The concentrations of corynetoxins U17a and H17a standard solutions were estimated by multiplication of the optical density measured at 260nm by their respective molecular weights (858 and 876) and dividing by the extinction coefficient 9650 at 260nm. The tunicamycin standard solution was prepared by weighing a commercial mixture of crystalline tunicamycins (purchased from Sigma Cat. T7765) and dissolving it in a known volume of 70% methanol in water, the concentration was confirmed by UV absorption at 260nm.
Development of ELISAs Two corynetoxin ELISAs were developed. One was an indirect, competitive ELISA whilst the other behaved as an indirect, non-competitive ELISA. Both were optimised and assessed for suitability for the detection of corynetoxins in fodder. Indirect, competitive ELISA This ELISA has sensitivity down to 0.4 parts per billion (40 picogram toxin/100µl /well) for authentic standards of corynetoxins or tunicamycins in assay buffer. The plates were first treated with 0.2 % glutaraldehyde in pH 9 carbonate buffer for 2 hours at 56oC. Excess glutaraldehyde was removed by washing with water before coating the plates with chemically modified tunicamycins. The glutaraldehyde treatment prepares the surface of the plates for coating with low molecular weight compounds, such as the modified tunicamycins, that do not readily coat on non-treated surfaces of ELISA plates. The plates were kept for 48 hours at 4oC before washing with 0.05% Tween 20 in saline to remove the unattached toxin derivative. Different amounts of the standard solutions of corynetoxins, tunicamycins and the individual corynetoxins H17a and U17a were made up in 1.0% bovine serum albumin (BSA), 0.05% Tween 20 in phosphate buffered saline (assay buffer) and were added to the wells in 100µl volumes along with the optimum dilution of anti-corynetoxin anti-serum in 50µl of assay buffer. After mixing the two solutions in the wells of the plates using an ELISA plate shaker, a two-hour period of competition was allowed for antibody to bind to the solid and liquid phase toxins. The unbound reagents and soluble toxin-antibody complex were 3
washed away with 0.05% Tween 20 in saline. The anti-corynetoxin antibodies bound to the surface of the wells were detected and quantitated by the addition of the appropriate dilution of anti-sheep IgG conjugated to horseradish peroxidase, purchased from Sigma (Sigma Cat. A3415). After one-hour incubation, the plates were washed and 3, 3’, 5, 5’- tetramethylbenzidine (Sigma Cat. T2885) substrate in pH 5.5 sodium acetate/citrate buffer was added and incubated for a further 15 minutes, before 50µl of 0.05 M sulphuric acid was added to stop the colour reaction. A comparison of the optical densities of the wells at 450nm for different amounts of corynetoxins, corynetoxin U17a, corynetoxin H17a, and tunicamycins is shown in Figure 1, and indicates that the ELISA detect each to the similar extent.
Optical density (450 nm)
1.20
1.00
0.80
0.60
0.40
0.20 10
100
1000
10000
Toxins (pg/well) Corynetoxins
Corynetoxin U17a
Corynetoxin H17a
Tunicamycins
Figure 1. Comparison of optical densities of the wells with different amounts of corynetoxins, corynetoxin U17a, corynetoxin H17a, and tunicamycin standards, showing competition for binding with 1/20000 diluted sheep 717 serum in glutaraldehyde-treated microtitre plates coated with 6 nanogram chemically modified tunicamycin/well. Novel indirect, non-competitive corynetoxin ELISA During previous experiments at CSIRO, a unique property of tunicaminyluracil toxins, such as corynetoxins and tunicamycins, possibly due to the presence of fatty acids chain in the toxin molecules, was noted. This led to the development of a novel type of corynetoxin ELISA. In the absence of BSA or when the concentration of BSA in the assay buffer was lower (< 0.25%) than the concentration in normal assay buffer (1.0%), the addition of sheep anti-corynetoxin antisera and corynetoxins or tunicamycins standards led to the adsorption of toxins and sheep anti-corynetoxin IgG on the surface of the wells of ELISA plates, pre-treated with 0.2 % glutaraldehyde. The quantitative increase in adsorption of toxins and sheep anti-corynetoxin IgG in the wells was shown by the increased enzymic activity of anti-sheep IgG conjugated to horseradish peroxidase (Figure 2), rather than decreasing activity expected in an indirect competitive ELISA.
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It is of interest to note that in Figure 2 for corynetoxin U17a, corynetoxin H17a and corynetoxin mixture with longer lengths of the fatty acid chain show higher binding strength (higher optical desity) than tunicamycin mixture, which has shorter lengths of fatty acid chain.
Optical density (450nm)
1.6
1.2
0.8
0.4
0 10
100
1000
10000
Toxins (pg/well) Corynetoxins
Corynetoxin U17a
Corynetoxin H17a
Tunicamycins
Figure 2. Standard curve produced by adding corynetoxins, corynetoxin U17a, corynetoxin H17a, and tunicamycins to the wells of a microtitre tray in the presence of anti-corynetoxin antibodies from sheep 237 (1/5000 dilution). The data were generated using 0.2% glutaraldehyde-treated microtitre trays. The graph showed increasing optical density indicating increasing attachment of anti-sheep IgG conjugated to horseradish peroxidase in wells corresponding to increasing amounts of added tunicaminyuracil toxins.
Relative detection capabilities of the two ELISA formats An extract of toxic annual ryegrass galls was subjected to HPLC, and one-ml/minute fractions were collected and assayed using the two corynetoxin ELISAs. Both ELISAs detect all the known corynetoxins. However, the indirect, competitive ELISA also detects an extra peak, eluting very early in the HPLC run, which is possibly a water-soluble precursor or metabolite of the corynetoxins.
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Extraction of corynetoxins from fodder Fodder samples were received with the compliments of Michael Mackie, Director of Gilmac Pty Ltd (W A), Malcolm May, Chairman, Balco Australia Pty Ltd (S A), and Kym Shardlow from Golden Plains Fodder Australia Pty Ltd (SA). Methods for extracting tunicaminyuracil toxins from fodder samples were tested using clean fodder spiked with known amounts of authentic, commercially available tunicamycins or by spiking with isolated bacterial galls enclosed in the husk of annual ryegrass seed (collected from Katanning, Western Australia). Selection of galls for spiking of fodder samples was randomised to lessen any effects of bias towards different sized galls. Commercially available tunicamycins, dissolved in 70% methanol in water, were added, one day before the extraction procedure, in volumes of 0.1 to 0.5 ml to 20 g of fodder in 500 ml laboratory bottles to achieve concentrations of tunicamycins from 50 to 500 ppb in the fodder. To assess extraction from the galls, samples of 20 g of fodder in 500 ml laboratory bottles were spiked with five bacterial galls and extracted with 200 ml of various extraction solvents. After 24 hours shaking, standing upright, in a Ratek orbital shaker at a speed setting of 8-10, 25 ml was removed from each bottle and centrifuged at 2000 rpm for 10 minutes. The clear supernatant was decanted into 2 x 10 ml centrifuge tubes (7 ml in each tube), and one Eppendorf tube (1 ml). The 2 x 7 ml were stored frozen at -20oC for future reference while the 1 ml samples was used for corynetoxin ELISA. Extraction solvents tested included alcohols, such as absolute methanol and ethanol, aqueous methanol and ethanol (70-80% in water), and aqueous extractants such as various molar concentrations of sodium hydroxide (0.01 M, 0.025M and 0.05M), detergents such as Tween 20 and Tween 80 (at 0.5%, 1.0% and 2.0%), and different concentrations of hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin (0.5%, 1.0% and 2.0%). The two different ELISA formats were found to possess different robustness to the matrix constituents extracted from fodder by the various extractants. In general, ethanol was found to be not as effective an extractant as methanol. Absolute methanol did not extract substances that interfered with either ELISA and it gave a very good recovery (80-100%) for the fodder samples spiked with known amount of pure tunicamycins. However, absolute methanol was not an effective extractant when the fodder samples were spiked with the bacterial galls enclosed in the husk of the annual ryegrass seed presumably because it was not effective in penetrating the enclosed galls. The sodium hydroxides and Tween-based solutions were also found to be ineffective in extraction of corynetoxins from fodder. Previous experiments at CSIRO have shown that aqueous methanol, such as 70-80% methanol in water, was very effective (up to 80% recovery by HPLC) in extracting corynetoxins from bacterial galls naturally enclosed in the husk of annual ryegrass seed. When fodder samples were extracted with 70-80% methanol and the extracts were diluted 1/10 in assay buffer, the two types of ELISA showed different effects. The indirect, competitive ELISA, showed no adverse effect due to the presence of extractives from the matrix. The novel, non-competitive adsorption ELISA however was significantly affected rendering it ineffective for assay of aqueous methanolic extracts of fodder. In addition, the use of poisonous methanol and the considerable volumes of the alcohol that will be required for extraction of large numbers of fodder samples raises concerns regarding cost and safety for its use with either ELISA format. Cyclodextrins form strong, water-soluble molecular complexes with corynetoxins and tunicamycins. CSIRO has previously successfully used different cyclodextrins as antidotes for the treatment of animals poisoned by corynetoxins and suffering from annual ryegrass toxicity. Cyclodextrins have also been used for capturing or binding of corynetoxins and tunicamycins. An HPLC method has shown that 1-2% aqueous methyl-β-cyclodextrin was as effective as 70-80% methanol in extracting corynetoxins from galls embedded in the husks of the annual ryegrass seed. The extraction efficiency was about 70-80%. The aqueous cyclodextrin solutions, at 1-2% (W/V), were subsequently shown to be effective as extractants for corynetoxin estimation using indirect, competitive ELISA format. CSIRO has applied these aqueous extraction methods to fodder samples. Preliminary assessment has also shown that the aqueous cyclodextrin 6
extractant was suitable for use with the R. toxicus ELISA. This would allow both the toxin and R. toxicus ELISAs to be used to assess the same extracts and this will facilitate comparison of these two ELISAs as indicators of the safety of fodder for livestock.
Validation of the indirect, competitive corynetoxin ELISA The indirect, competitive corynetoxin ELISA, that was more robust and showed no adverse effect due to the presence of extractives from the matrix when fodder samples were extracted with 80% methanol or 1% to 2% methyl-β-cyclodextrin, was validated against a confirmatory HPLC assay before applying to 113 fodder samples. Reagent grade, deionised water (200 µl) was added to each of thirty tubes containing 6 bacterial galls still within the seed husk. After 1 hour, the swollen and softened bacterial galls were crushed using a polyethylene rod (plunger from 1ml syringe with the bottom end formed into a blunt conical point). Methanol (800 µl) was added, washing down the rod in the process. The samples were shaken for 20 hours, centrifuged and the supernatants removed. The supernatants were then assayed by HPLC and by indirect, competitive corynetoxin ELISA (Table 1).
Results HPLC and indirect, competitive corynetoxin ELISA The mean corynetoxin content (µg/sample) of six crushed galls extracted with 80% methanol was 16.00 (± 3.98 SD) when assayed using HPLC and 15.10 (± 4.01 SD) by indirect, competitive corynetoxin ELISA (Table 1, Figure 3). Paired data were analysed by using t-Test: Paired Two Sample for means. The data showed that means of corynetoxin content (µg/sample) assayed by HPLC and by ELISA were not significantly different: P (T
