Detecting Ergot Alkaloids by Immunoassay - ACS Symposium Series

Chapter 17

Detecting Ergot Alkaloids by Immunoassay

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Richard A. Shelby Department of Plant Pathology, Auburn University, Auburn, A L 36849

The ergot alkaloids are seldom a problem in grain products destined for human consumption, due to the relative ease of removing sclerotia of Claviceps from most cereal grains which are hosts for this genus of toxigenic fungus. Animal feeds, on the other hand are often contaminated with ergot alkaloidsfromClaviceps, as well as from fungi of the genus Acremonium, which are endophytic in many pasture grasses worldwide. We have employed two strategies in the development of polyclonal and monoclonal antibodies against these alkaloids. Depending on the point of conjugation with the carrier protein, antibodies can be produced which are specific for a narrow range of target moieties, or antibodies which are more general and react with a broad range of ergot alkaloids are produced. These antibodies are mainly used in detection in common ELISA formats.

The ergot alkaloids differ from other natural food toxicants in several important respects. This mycotoxin has probably been responsible for cases of human toxicosis since prehistoric times, and is certainly one of the few for which mass human poisonings can be traced to a known specific class of mycotoxin from the historic record. "Holy fire" or "St. Anthony's fire" was the name given to the gangrenous ergotism which plagued medieval Europe until consumption of ergot sclerotia was recognized as the cause of the illness (1). The production of sclerotia from infected seed of the host grain is another distinctive feature of this fungal plant pathogen. Unlike other mycotoxigenic fungi, the genus Claviceps typically produces enlarged sclerotia in place of the grain, which are dark in color, and can be easily separated from the healthy grain. At least 10 species of Claviceps are recognized on 50 genera of grass hosts (Table I). Finally, the ergot alkaloids exhibit a broad range of pharmacologic effects. Rather than being acutely toxic or carcinogenic, as many mycotoxins apparently are, these compounds can actually be beneficial pharmaceutical

0097-6156/%/0621-0231$15.00/0 © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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IMMUNOASSAYS FOR RESIDUE ANALYSIS

compounds when administered properly. For this reason the ergot drugs are still produced by growing the pathogen on rye, harvesting sclerotia, and extracting the crude ergot compounds. These compounds can be purified and then modified to produce other semisynthetic forms. Table I. Fungi Known to Produce Ergot Alkaloids Pathogen

Host

Acremonium coenophialum Acremonium 3 spp. Epichloe typhina Claviceps 10 spp.

Aspergillus Pénicillium Balansia Sphacelia Hypomyces Phycomyces Rhizopus

Reference

Festuca arundinacea

(2)

5 species in 4 genera

(3)

8 spp. in 4 genera

(3)

200 spp, 50 genera (all Poaceae)

(4)

Various

(1)

Like many other mycotoxins, the ergot alkaloids exist as a large family of compounds, the various forms of which differ widely in toxicologic or pharmacologic effect, depending upon the substitutions on the D ring of the 5-membered ergoline ring structure (Figure 1). There are now about 100 members of this family, including semisynthetic drugs. They range in pharmacologic activity from the semisynthetic A^A^-diethyl-D-Lysergarnide (LSD-25), one of the most powerful psychomimetics ever produced, to ergotamine, used to treat migraine headache. The range of pharmacologic activity produced by modifications of this simple ergoline ring structure may still produce fascinating insights into basic endocrinology and neurochemistry. A n exhaustive review of ergot chemistry can be found in Berde and Schild (5). As mycotoxins, this group of compounds rarely poses a threat to human health, owing to current sanitation procedures in grain processing. The ergot alkaloids are common in grain products, particularly rye (6), but at levels which probably have minimal negative health potential. They do, however, pose a significant health risk to animals. Grazing livestock can frequently ingest Claviceps sclerotia in the infected grain directly from the field on any of the 50-odd genera of grasses which are potential hosts for this fungus. Grain which has been refused for human consumption could potentially be used as livestock feed, or when used directly on the farm where

Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

17. SHELBY


COMPOUND ERGONO VINE LYSERGIC ACID ERGOSINE ERGO VALINE ERGOTAMINE FESTUCLAVINE ELYMOCLAVINE

DOUBLE BOND

R CONHC (CH ) C H OH COOH CYCLOL ALA-PRO-LEU CYCLOL ALA-PRO-VAL CYCLOL ALA-PRO-PHE β- CH C H OH 3

3

2

2

9-10 9-10 9-10 9-10 9-10 8-9 8-9

Figure 1. Ergot alkaloids generalized structure.


233

234


it is produced without appropriate testing. Another common form of ergotism in livestock arises from ingestion of pasture grasses infected with endophytic fungi, predominantly Acremonium coenophialum in tall fescue, and Acremonium lolii in perennial ryegrass. The symptoms produced in animals ingesting ergot alkaloids typically include poor weight gain, poor milk production in lactating animals, various reproductive disorders, and in extreme cases, the classical gangrenous loss of extremities such as feet, tails, and ears. In the cases of animal intoxication, like those of ergotism in humans, the effect is produced only when there is constant dietary intake of the contaminated food source as is the case when animals are confined to an infected paddock or pasture. Methods of Detection Several methods are available for detecting ergot alkaloids. Perhaps the oldest is the colorimetric assay based on /7-dimemylaminobenzaldehyde (7). This chromagen, also, can be used as a spray reagent with thin-layer chromatography (TLC), where the color of the spot developed is characteristic of the alkaloid (8). The ergot compounds that have a double bond in the 9,10 position are strongly fluorescent (Figure 1), making detection in solution or on a T L C plate relatively simple. Fluorescence detection is also nondestructive, making it possible to harvest pure alkaloid from the T L C plate or liquid chromatography outflow. The ergot alkaloids not having the 9,10 double bond, usually, can be detected by ultraviolet absorbance of the ergoline ring structure. Fluorescence and U . V . absorbance facilitate high-performance liquid chromatography (HPLC) (9-11). Tandem mass spectrometry (MS/MS) also has been used to detect and identify ergot alkaloids in fescue (2). In a diagnostic laboratory or field situation, the need often arises to survey large numbers of samples. In these situations, it is more important that samples contaminated with trace amounts of the target compound be identified, rather than quantitative accuracy. It is this situation for which immunoassay techniques are ideally suited. We typically have available more than one method to detect a target compound. This enables us to select a method which is most economical and efficient, as well as providing a methods check whereby analysis by one method can be compared with that of a second or third method. In the case of ergot alkaloids, for example, we can use T L C , HPLC, and immunoassay. Each method yields a different type of data, useful in its own way. H P L C data is typically the most quantitatively accurate, since each moiety in a complex mixture is separated, detected, integrated by computer, and compared with analytical standards. The nature of the procedure, however, demands a high degree of training of personnel, expensive equipment, and utmost attention to detail. In practical terms, without an automated HPLC, only about 15 samples per day can be analyzed at a cost of about $25.00 per sample. The immunoassay format requires less investment in equipment, training, and time per sample. We can analyze hundreds of samples per day at a fraction of the cost per sample. The trade-off comes in the sacrifice of quantitative accuracy of the data. There are inherent sources of error in all immunoassay methods, particularly when the target compound is in a complex background of related molecular structures.


17. SHELBY


235

Early Immunoassays The ergot alkaloids were one of the first mycotoxin groups for which immunoassay methods were developed. The early interest in radioimmunoassay (RIA) methods stems from the importance of ergot alkaloids as drugs (12-16). These methods were developed for use in pharmacokinetic studies where picogram amounts of ergot drugs were detected in plasma and other biological fluids. These early immunoassays pointed out the effectiveness of the competitive immunoassay in adding quantitative precision to the assay. Another important prior discovery was the observation by Arens and Zenk (16) that the point of conjugation on the ergot alkaloid molecule results in antibodies with dramatically different specificities. These early successes at producing antibodies against ergot alkaloids suggested that it would be practical to develop our own antibodies and immunoassays to detect ergot compounds in agricultural commodities. They also suggested conjugation strategies which would result in immunogens that should produce antibodies of different affinities and specificities. Ergovaline and Endophytes Our laboratory serves primarily a diagnostic function to assist livestock owners, veterinarians, and the seed industry in identifying fields which may have endophyte toxicosis problems. This has been done mainly by micropscopic examination of plant and seed material for evidence of fungal hyphae (17). With the discovery of ergovaline in endophyte-infected tall fescue (2) another potential diagnostic tool became available. If ergovaline proved to be the "endophyte toxin", then levels of this compound should be a good indicator of toxic potential in the pasture. A reliable H P L C method for analysis of ergovaline in fescue was available, and this quickly became a standard procedure in our lab, but the previously discussed limitations of H P L C make immunoassay for this mycotoxin an attractive alternative. Research samples submitted to the lab sometimes consist of large numbers of individual samples, sometimes less than 1 g dry weight. Plant breeders, for example, might wish to determine the toxic potential of large numbers of individual plants from a greenhouse experiment, where the plants tend to be smaller. Our goal was to develop an immunoassay which would enable us to identify toxic plant material in samples collected by researchers and the general public. The development of specific anti-ergot alkaloid antibodies was proven technology, but could be improved upon by the use of enzymes rather than radioisotope labels in the assay. This would avoid the inherent problem of isotopes being a hazardous waste, and result in an assay using a chromagen which could be interpreted visually. The enzyme-linked immunosorbent assay (ELISA) has essentially replaced the RIA for these reasons. The technology of monoclonal antibodies also would adds specificity and reproducibility to an ELISA. A problem associated with production of antibodies to ergovaline was the very limited amount of pure ergovaline available. Milligram quantities were obtained from Sandoz (Basel, Switzerland) but it was practical to use this only for standards in chromatography. This meant that our initial attempts to produce antibodies had to be with structurally related compounds. Because of the pharmacological importance of


236


ergot drugs, several other similar compounds are readily available. We chose ergotamine because of its structural similarity to ergovaline. Ergotamine Polyclonal Antibody Our first attempt to produce anti-ergot antibodies were essentially a duplication of the work of several predecessors (18). To make the immunogen, ergotamine was linked to the carrier protein bovine serum albumin (BSA) at the indole nitrogen (Figure 1) by the Mannich reaction using glutaraldehyde as the linking agent (Figure 2). A second conjugate was made utilizing ovalbumin (OVA) as the carrier for subsequent use in coating polystyrene microtiter plates in the ELISA. Estimation of conjugation efficiency or molar substitution ratios can be made by measuring the amount of unbound hapten after completion of the reaction. This is removed by dialysis before use. A second relatively simple way to estimate conjugation efficiency is to run the conjugate in the T L C system for these alkaloids, and view the plate in U . V . light or spray with Erlich's reagent. Unbound ergotamine will move to the usual position on the plate as indicated by standards, while the conjugate will remain at the origin of the plate. Relative size of the spots is an indication of conjugation efficiency. We estimated the molar substitution ratios to be 7.7 for B S A and 1.3 for O V A . This reflects the relative abundance of lysine residues to which the aldehyde links the hapten in the two proteins. We measured the protein in the conjugate by colorimetry and adjusted the dosage to approximately 1 mg per injection. Rabbits were immunized following the general protocols found in Harlow and Lane (19). Titers were measured by competitive inhibition (CI) ELISA (Table II). Serum titers were acceptable after the second boost, and a single lot of serum was chosen for purification and testing. Using the competitive inhibition protocol, with a second antibody conjugate (Goat-antimouse-peroxidase) makes purification of the antiserum unnecessary. We have found that simple ammonium sulfate precipitation is sufficient. Using commercially available second-antibody enzyme conjugates assures a reproducible enzyme reaction if purchased from a reliable vendor with adequate quality control. As expected, this antibody showed the greatest affinity for the target alkaloid, ergotamine, and ergot alkaloids with related structures (Table III). It appears that the 2' position in the molecule is generally not involved in recognition (Figure 1). However, it is readily apparent that the cyclol-peptide portion of the molecule is the site mainly involved in recognition. A rule of immunology was borne out: the region of the hapten involved in recognition is distal to the point of conjugation. Fortunately, many of these alkaloids are produced by Claviceps (20) and the antibody could be used in this assay. Unfortunately, the antibody had little or no affinity for ergovaline, and was unsuitable for analysis of endophyte-infected fescue. Ergonovine Monoclonal Antibody As mentioned above, several previous researchers noted that using other reactive sites on the ergot alkaloid molecule, resulted in differing reactivity spectra of the resultant antibodies. Arens and Zenk (16) found that using lysergic acid as the hapten, linking B S A at the carboxyllic acid moiety at the C8 position resulted in an antibody which would react with simple lysergic acid derivatives, clavines, and peptide alkaloids. We


17. SHELBY


237

Table II. CI-ELISA Generalized Protocol

1.

ELISA plates = Dynatech Immulon 4.

2.

Coat with hapten-carrier conjugate 1/1000 in 9.6 carbonate buffer, 60 min 30 °C.

3.

Wash 5x phosphate buffered saline + 0.05% tween 20 (PBST), no wait.

4.

Sample preparation. Dilute standards from lmg/mL stocks in PBST. Start with 1000 ppb, or dilute standards in sample extract of plants or seeds. Prepare samples by grinding to pass a 2 mm screen. Weigh out 1 g in disposable plastic cup. Add 10 mL PBST. Allow to sit at room temperature 30-60 min while plate is coating or overnight at 5 °C. Pipette supernatant into ELISA well (50 microliters).

5.

Add antibody (50 μ ί ) diluted in PBST. Optimum dilution varies and must be determine for each Ab lot. Sit 15 min at room temperature. Wash 5x.

6. 7.

Add goat anti-mouse peroxidase conjugate 1/1000 in PBST at room temperature and incubate for 15 min.

8.

Wash 5x.

9.

Chromagen = orthophenylinediarnine dihydrochloride (OPD) 1 mg/mL in urea peroxide buffer at room temperature for 15 min is average. Stop reaction with 3 M sulfuric acid.

10.

Read on an ELISA plate reader at 490 nm.

11.

Calculate regression equation for standard curve. Solve unknowns for ppb.


238


Table III. Cross-reactivity of Poly- and Monoclonal Antibody

50% inhibition (ng/mL)

Compound

Polyclonal

Ergotamine Ergotamimne Ergostine Ergocristine Ergostinine Ergosinine Ergosine Ergoptine a-ergokryptine Ergovaline Ergonine Festuclavine Setoclavine Ergonovine Ergocornine Elymoclavine Rugulovasine Pyroclavine Agroclavine LSD Lysergic Acid Costaclavine L-tryptophan

0.99 3.5 3.7 4.4 12.0 38.3 50.6 238.0 345.0 1533.0 1937.0 2995.0 3121.0 3720.0 3775.0 4361.0 7338.0 17151.0

-

-

a

Monoclonal

129.0 1129.0 3361.0 545.0 3031.0 216.0 66.0 191.0 1863.0 71.0 533.0 181.0 72.0 0.05 446.0 59.0 >10,000.0 560.0 40.0 167.0 337.0 3879.0 >10,000.0

Concentration of compound causing a 50% reduction in ELISA values when compared to buffer-only controls.


17. SHELBY


239

used ergonovine by first making the hemisuccinate following the procedure of Stason, et al. (21) (Figure 3). We also decided to attempt a monoclonal antibody, since that capability had recently been made available to us. A n additional change was to make a second coating conjugate, using poly-l-lysine as the carrier. This resulted in a much higher substitution ratio than could be obtained with natural proteins. The production of the monoclonal antibody proceeded along the same general protocol used above, with some exceptions. Mice were tested for polyclonal titers using the same CI-ELISA as before, and the mouse with the highest titer was chosen for boosting and fusion using a standard protocol (23). Next comes the arduous task of selecting the best antibody-producing clones. Important criteria to consider are: 1) monoclonality of the hybridoma, 2) vigorous growth of the hybridoma, 3) high rate of antibody production, and 4) specificity of the antibody for the target. Of secondary consideration is the globulin subclass produced by the cell line. The above criteria are assured by scrupulous attention to detail, including keeping accurate records of cell lineage when replating cells. If one is not especially ruthless in discarding inferior cell lines, the exponential progression of growing cells can quickly be overwhelming. Before proceeding with a fusion, it is imperative that the screening protocol be thoroughly tested to ensure that the test (in our case, CI-ELISA) will correctly identify targe-specific clones. Using the polyclonal sera from tail bleedings of the mice accomplishes this. We selected the best clone from several promising hybridoma lines, based on the criteria mentioned above. The resulting antibody works well in the CI-ELISA when the source is unpurified hybridoma supernatant solutions. Appropriate concentration of hybridoma supernatant solutions must be determined empirically for each hybridoma lot derived from 24-well plates or from bulk production. Maximum antibody production is obtained from ascites fluid. The selected clone, EN9F10, demonstrateed a wide spectrum of anti-ergot alkaloid activity (Table III) which makes it suitable for testing not only Claviceps infected material, but also Acremonium infected fescue as well. The enhanced sensitivity of this assay when compared to the polyclonal assay derives from several factors. The cross-reactivity of the antibody makes more target available to which the antibody can bind. This is due to the fact that both Claviceps and endophyte infections result in more than one member of the ergot alkaloid family being produced. In the case of Claviceps, at least 6 different alkaloids and their epimers are being produced (6) and this would not include clavines and nonfluorescent alkaloids. Another reason for enhanced sensitivity is probably the higher substitution ratio of the PLL-conjugate. This makes for a greater affinity of the antibody for the polystyrene plate, resulting ultimately in a faster, darker chromagen reaction. We calculated that, with this antibody in the CI-ELISA, we could detect one Claviceps sclerotium in 20 kg of grain. Practical Considerations Several factors contribute to quantitative inaccuracy in this assay. The same may also apply to other types of immunoasssays. First, quantitative inaccuracy resulting from the fact that the target alkaloids are a family or population of cross-reactive antigen targets which are never present alone in naturally occurring sources. Since each antigenic target has a slightly different coefficient of cross-reactivity (50% inhibition


240


N H NH2 NH2 LYS LYS LYS

ERGOTAMINE-BSA CONJUGATE

2

1

1

1

BOVINE SERUM ALBUMIN (BSA)

Figure 2. Cross linking of Ergotamine to BSA via glutaraldehyde.

ERGONOVrNE

SUCCINIC ANHYDRIDE

0

0

VY.

E N - C - N H - ÇH-CH3 H-Ç-H

HEMISUCCINATE

E N - C - N H - CH-CBj

2

Ί

OH

ο

V A O

OH

EN-C-NH-ÇH-CHj

ςΉ

2

fl

CARBODIIMIDE

ΝΗ

\

LYSINE

LYSINE

ALBUMIN OR POLY-L-LYSINE

HAPTEN-CARRIER CONJUGATE

Figure 3. Conjugation of Ergonovine and PLL.


17.

SHELBY


241

ratio), this makes quantitative analysis difficult. Competitive inhibition in an ELISA will be the sum of each individual reactant and the standard curves generated from a single antigen will not apply to the mixture. Since the ratios of alkaloids in the mixture cannot be predicted, a standard curve produced by an artificial mixture of alkaloid cannot be generated. The best approximation would be to select a standard antigen target and express the data in units of that antigen. For example, we use ergonovine, and express the results as parts per billion (ppb) ergonovine in solution, understanding, of course, that this is erroneous. A l l antibodies, even monoclonal antibodies, may be somewhat cross-reactive, so all ELISA tests of a naturally occurring compound in a natural substrate will suffer to some extent from this error. Mycotoxins, being at the end of a biosynthetic pathway, and suffering from bio- and other degradation, are especially liable to this error. The other flaw contributing to quantitative inaccuracy is the problem of insolubility. Since antibodies have clear optima of ionic strength, pH, temperature and other parameters, they function optimally in an aqueous solution of phosphate buffered saline. Any deviation from these optima will result in reduced affinity of the antibody and reduced accuracy of the immunoassay. Many of the targets of immunoassays are poorly soluble in aqueous solutions, so other solvents like methanol or acetonitrile must be used. The ergopeptides fall into this category. While some of the clavines and simple lysergic acid derivatives are more hydrophilic, most organic solvents will denature the antibody. ELISA tests for hydrophobic compounds must use dilute mixtures of denaturing solvents. Barna-Vetro et al. (24) suggest that alcohol or acetonitrile concentrations should not exceed 10% in the antibody reaction mixture. We have observed the same in our laboratory. The quantitative inaccuracy of the CI-ELISA using EN9F10 is acceptable if the test is used for screening purposes to eliminate samples which are not contaminated, and require no further testing. Those samples indicated as positive in the assay can be further scrutinized by more accurate methods, such as HPLC. Acknowledgments The author wishes to thank Dr. Virginia Kelley for her invaluable assistance and immunological expertise in the development of the antibodies described herein. Literature Cited 1. 2. 3.

4. 5.

Rehacek, Z.; Sajdl, P. Ergot Alkaloids; Academia Press: Prague, Czech Republic, 1990. Yates, S. G.; Plattner, R. D.; Garner, G. B. J. Agric. Food Chem. 1985, 33, 719-722. Siegel, M. R.; Latch, G. C. M.; Bush, L. P.; Fannin, F. F.; Rowan, D. D.; Tapper, Β. Α.; Bacon, C. W.; Johnson, M. C. J. Chem Ecol. 1990, 16, 3301-3315. Farr, D. F.; Bills, G. F.; Chamuris, G. P.; Roseman, A. Y. Fungi on Plants and Plant Products in the United States; APS Press: St. Paul, MN, 1989; p 632. Ergot Alkaloids and Related Commpounds; Berde, B.; Schild, H. O., eds.; Handbook of Experimental Pharmacology; Springer Verlag: Berlin, Germany, 1978, Vol 49.


242 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

IMMUNOASSAYS FOR RESIDUE ANALYSIS Scott, P. M.; Lombart, G. Α.; Pellaers, P.; Bacler, S.; Lappi, J. J. AOAC. 1992, 75, 773-779. Allport, N. L.; Cocking, T. T. Yearbook Pharm. 1932, 5, 341-46. Svendsen, A. B.; Verpoorte, R. Chromatography of Alkaloids. Chromatography Library 23A; Elsevier: Amsterdam, 1983; pp 388-389. Heacock, R. Α.; Langille, K. R.; MacNeal, J. D.; Frei, R. W. J. Chromatogr. 1973, 77, 425-430. Scott, P. M.; Lawrence, G. A. J. Agric. Food Chem. 1980, 28, 1258-1261. Yates, S. G.; Powell, R. G. J. Agric. Food Chem. 1988, 36, 337-340. Van Vunakis, H.; Farrow, J. T.; Gjika, H. B.; Levine, L. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 1483-1487. Taunton-Rigby, Α.; Sher, S. E.; Kelley, P. R. Science 1973, 181, 165-166. Rosenthaler, J.; Munzer, H. Experentia. 1975, 32, 234-235. Schran, H. F.; Schwartz, H. J.; Talbot, K. C.; Loeftler, L. J. Clin. Chem. 1979, 25, 1928-1933. Arens, H.; Zenk, M. H. Planta Medica. 1980, 39, 336-347. Shelby, R. Α.; Dalrymple, L. W. Plant Disease. 1987, 71, 783-786. Shelby, R. Α.; Kelley, V. K. J. Agric. Food Chem. 1980, 38, 1130-1134. Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1988; pp 92-120. Porter, J. K.; Bacon, C. W.; Plattner, R. D.; Arrendale, R. F. J. Agric Food Chem. 1987, 35, 359-361. Stason, W. B.; Vallotton, M.; Haber, E. Biochim. Biophys. Acta. 1966, 133, 582-584. Shelby, R. Α.; Kelley, V. K. J. Agric. Food Chem. 1992, 40, 1090-1092. Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY, 1988; pp 139-243. Barna-Vetro, I.; Gyongyosi, Α.; Solti, L. Appl. Environ Microbiol. 1994, 60, 729-731.

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Detecting Ergot Alkaloids by Immunoassay - ACS Symposium Series

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