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Detection of Ergot Alkaloids in tallfescue by competitive immunoassaywith a monoclonal antibodyRichard A. Shelby a & Virginia C. Kelley aa Departments of Plant Pathology, and Botany andMicrobiology, Alabama Agricultural Experiment Station,Auburn University, AL, 36849, USAPublished online: 16 Sep 2008.

To cite this article: Richard A. Shelby & Virginia C. Kelley (1991): Detection of Ergot Alkaloidsin tall fescue by competitive immunoassay with a monoclonal antibody, Food and AgriculturalImmunology, 3:3-4, 169-177

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Food & Agricultural Immunology (1991) 3, 169-177

Detection of Ergot Alkaloids in Tall Fescue byCompetitive Immunoassay with a Monoclonal Antibody

RICHARD A. SHELBY AND VIRGINIA C. KELLEY

Departments of Plant Pathology, and Botany and Microbiology, AlabamaAgricultural Experiment Station, Auburn University, AL 36849, USA

(Received for publication 19 July 1991)

Ergonovine was coupled to human serum albumin by a modification of the carbodi-imide procedure. The conjugate was used to immunize mice, one of which developedspecific anti-ergonovine antibodies as measured by a competitive indirect (CI) enzyme-linked immunosorbent assay (ELISA). Lymphocytes from this mouse were used toproduce a hybridoma cell line which continued to produce specific anti-ergonovinemonoclonal antibody (MAb); the MAb was then used to develop an assay for ergotalkaloids associated with fescue (Festuca arundinacea) infected with the endophyticfungus Acremonium coenophialum. In this CI-ELISA, the 50% inhibition value of theMAb by ergonovine was approximately 0.5 ng ml-1. The MAb recognized clavine,ergopeptine and lysergic acid derivatives. In tall fescue plants, the assay identifiedpastures with infection percentages as low as 3% and could be used to determineinfection in a single plant, using as little as 0.5 g of material. Infection in seed wassimilarly detectable at infection levels as low as 4%. The assay offers significantadvantages over diagnosis by microsopy or high-performance liquid chromatography interms of time, cost and special conditions required.

Keywords: ergometrine, ergovaline, fescue toxicosis

INTRODUCTION

The ergot alkaloids are toxic metabolites produced by a number of parasitic andendophytic fungi, most notably by members of the genus Claviceps. This class ofalkaloids is represented in nature by a large family of compounds related to lysergicacid. We became interested in these alkaloids after their discovery in tall fescue(Festuca arundinacea Schreb.) infected by the endophytic fungus Acremoniumcoenophialum Morgan-Jones and Gams, and their implication as a possible causeof fescue toxicosis (Porter et ah, 1979). From the /icremon/Mm-infected fescue anumber of ergot alkaloids have been identified: chanoclavine, ergosine and its epimer(Porter et ai, 1979), ergotamine, ergosine, ergovaline, ergostine, ergoptine, ergonine,ergocrystine, ergocornine (Yates et ah, 1985), and recently lysergic acid amide

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1 7 0 R. A. SHELBY & V. C. KELLEY

(Petrosky & Powell, 1991). The present paper describes a monoclonal antibody used todevelop an enzyme immunoassay. This assay offers more sensitive detection of theergot alkaloids of endophyte-infected fescue than enzyme assays previously reported.

MATERIALS AND METHODS

Preparation Of Protein ConjugatesFor use as a conjugate to coat polystyrene microtiter plates, D-lysergic acid (LA, SigmaL-5376, Sigma, St Louis, MO) was conjugated to poly-L-lysine hydrobromide (PLL,Sigma P-1274, Sigma, St Louis, MO) by a modification of the method of Loeffler &Pierce (1973) which links the e-amino groups of the PLL to the carboxylic acid portionof LA using l-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC,Pierce 22980H, Pierce Chemical Co., Rockford, IL). LA (2 mg was dissolved in 1-67ml water containing 10% methanol to which 10 mg PLL dissolved in 250//I water wasadded while stirring constantly. This was followed by the addition of 3 mg W-hydroxysulfosuccinimide (Sulfo-NHS, Pierce 2451 OH, Pierce Chemical Co., Rockford, IL;Staros et al., 1986) and 20 mg EDC in 167 fi\ water. The pH was adjusted to 7-0 with0-1 M-NaOH and the mixture was stirred in darkness for 30 h at room temperature.The reaction was stopped by adjusting the pH to 4-5 with 1 M-sodium acetate. Themixture was dialyzed (MW cut-off=10 000) at 4°C for two days against phosphate-buffered saline (PBS), then against distilled water with frequent changes until therewas no visible fluorescence in the dialysis water. The dialysate was dispensed into 0-5aliquots and stored at — 20cC.

For immunizing mice, ergonovine (EN) was conjugated to human serum albumin(HSA) by a modification of the method of Reddick et al. (1991). The hemisuccinate ofergonovine was prepared by incubating ergonovine maleate (Sigma E-6500) withsuccinic anhydride (Sigma S-7626) in water at pH 6-8 with constant stirring indarkness at room temperature. The pH was maintained with 5% (w/v) NaOH. Thecourse of the reaction was monitored by reverse-phase high-performance liquidchromatography (HPLC) with fluorescence detection (method of Rottinghaus et al.,1990). A maximum succinate-to-free-ergonovine ratio (43/54) was observed after threedays. EN-hemisuccinate was isolated by loading the mixture onto a Waters C-18Sep Pack (Waters Chromatography, Milford, MA) and eluting in 25% methanol, whichwas subsequently removed by lyophilization.

EN-hemisuccinate was then conjugated to HSA (Sigma A-8763) as follows. HSA (4-5mg) was diluted in 2-25 ml of 0-1 M-PBS (pH 7-4). To the protein was added 3 mg ofEN-hemisuccinate and the pH was adjusted to 7-2 with 1 M-NaOH. One milliliters of a20 mg ml~' solution of EDC was added dropwise with stirring. Ten milligrams ofSulfo-NHS was then added and the pH again adjusted to 7-2. The mixture was stirredat room temperature in the dark overnight and dialyzed extensively against water andfinally against PBS. By measuring free EN-hemisuccinate in the dialysate, the courseof the dialysis was monitored and the degree of conjugation estimated. Based on thismethod, the molar substitution ratio of EN to HSA was estimated to be 62:1. A similarconjugate of EN was made using glutaric anhydride rather than succinic anhydride asthe linking group.

Immunization of MiceFor primary immunization, the EN-HSA conjugate (succinate-linked), adjusted to 1mg HSA ml"1, was mixed with an equal volume of Freund's complete adjuvant bysonication until a stable emulsion was obtained. Each of three B ALB/c mice receivedapproximately 300 fi\ of the emulsion, divided into several subcutaneous and intra-peritoneal sites. After five weeks the mice were boosted with the same volume of an

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DETECTION OF ERGOT ALKALOIDS IN TALL FESCUE 1 7 1

EN-HSA conjugate (glutarate-linked) emulsified with Freund's incomplete adjuvant,administered similarly. Nine weeks after the second immunization, results of CI-ELISA (see below) of tail bleedings demonstrated that one mouse had anti-ENantibodies. This mouse was selected for hybridoma production. Twelve weeks follow-ing the second immunization and three days prior to hybridoma fusion, the mousewas given a final intravenous booster immunization of approximately 100 /zg EN-HSA(glutarate-linked) without adjuvant.

Monoclonal Antibody ProductionSpleen cells from the immunized mouse were fused with myeloma cells (X63-Ag8-653)in active logarithmic growth, using polyethylene glycol (Harlow & Lane, 1988). Thecell suspension was placed in RPMI-HAT medium and seeded in 10 96-well cultureplates containing feeder macrophages. After one week of incubation at 37°C in a CO2incubator, virtually all wells on all plates had visible hybridoma growth and weresubsequently screened by CI-ELISA. Of approximately 900 wells tested, fewer than 30showed any significant antibody production and only four had both high levels ofantibody and high competitive inhibition values. Three of these four cell lines retainedthese characteristics upon cloning, and were recloned, retested, and expanded. Super-natants were collected and hybridoma cells were frozen and stored in liquid nitrogen.The isotypes of the antibodies were determined using a double diffusion isotyping kit(ICN Immunobiologicals 64-690-1 (Costa Mesa, CA)). One clone (EN9F10) wasdetermined to be of the IgG2b isotype and the others were IgGl isotype. EN9F10was chosen for subsequent evaluation.

Competitive Indirect (CI) ELISANinety-six-well ELISA plates (Immulon 4, Dynatech corporation, Chantilly, Virginia)were coated with 100 fil of a 1:4000 dilution (approximately 825 ng ml"1) of theLA-PLL conjugate in 005 M-sodium carbonate buffer, pH 9-6. Plates were incubatedat room temperature for 30 min and washed briefly five times with PBS+0-05%Tween 20 (PBST) using a hand-held wash bottle. For the competitive inhibition step,the alkaloid solution to be tested, which varied with the test and sample type (seebelow) was added in a volume of 50 fil. Antiserum in the form of hybridomasupernatant was diluted 1:200 in PBST plus 1%, w/v, non-fat dry milk (PBSTM) and50 /zl was added to the well. Following incubation at room temperature for 15 min, theplates were washed as above with PBST. Bound antibody was measured usingcommercial goat anti-rabbit IgG (H plus L) peroxidase conjugate (Bio-Rad Labora-tories, Richmond, CA, 170-6515), diluted 1:1000 in PBST. After incubation at roomtemperature for 15 min, the plate was washed as before with PBST. To each well, 100/d of the chromogen o-phenylenediamine dihydrochloride, OPD (Sigma P-8287)dissolved at 1 mg ml"1 in 100 mM-sodium citrate buffer pH 5-0 plus 0-003% hydrogenperoxide, was added. Color development at room temperature was stopped after 15min with 50 [A per well of 3 M-sulfuric acid. Optical density was measured at 490 nmusing a Dynatech MR 580 reader.

Cross-reactivityAuthentic standards of alkaloids for spiking, cross-reactivity, and controls in quantita-tive CI-ELISA were dissolved in methanol as 1 mg ml"1 stock solutions and held at— 20°C. To calculate cross-reactivity, stock alkaloid solutions were diluted to 1 ngml"1 in PBST and serial 1:2 dilutions were made directly on the horizontal axis of theELISA plate.

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1 72 R. A. SHELBY & V. C. KELLEY

Fescue Plant SamplesFescue plant material consisting of panicles and culms at anthesis were collected frompastures of the Alabama Agricultural Experiment Station and examined by micros-copy for the presence of the endophyte (Shelby & Dalrymple, 1987). For bulkedsamples, approximately 100 culms with panicles were dried at 60°C for 8 h andground in a laboratory mill to pass a 2 mm screen. Ergot alkaloids were determined byHPLC using the method of Rottinghaus et al. (1991). For the CI-ELISA, dried groundplant material was weighed into a disposable plastic cup to which was added ten timesas much PBST (by volume), this then being stirred briefly. This was allowed to standfor 30 min at room temperature. The liquid portion of the sample was then pipetteddirectly into LA-PLL-coated wells. Fifty microliters of liquid were replicated in fouradjacent wells for each sample. Monoclonal antibody was added and the remainder ofthe assay was conducted as described previously. For fresh plant material, single culmswere those described as above and sap expressed by means of a roller press into adisposable plastic cup. Fifty microliters of fresh sap were pipetted directly into theplate wells, and the remainder of assay was conducted with replications exactly asbefore.

Fescue SeedTwelve seed lots previously tested for endophyte infestation percentage by microscopywere chosen to include a range of infestation levels from 0% to 100%. Grinding andsubsequent CI-ELISA protocol was identical to that for dried plant material.

TABLE 1. Cross-reactivity of anti-ergonovine mono-clonal antibody EN9F10

Compound

ErgonovineAgroclavineElymoclavineErgosineErgovalineSetoclavineErgotamineLSDFestuclavineErgoptineErgostinineLysergic acidErgocornineErgonineErgocrystinePyroclavineErgotaminineA-ergokryptineErgostinineErgostineCostaclavineRugulovasineL-Tryptophan

50% inhibition"

ng ml"1

00464059667172

129167181191216337446533545560

11291863303133613879

>10000>10 000

flM

0-000140170-230-120-130-280-220-520-720-340-391-260-800-970-892-221-943-245-095-64

15-39>37>49

"Concentration of compound causing a 50% reductionin CI-ELISA values when compared to buffer-onlycontrols.

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DETECTION OF ERGOT ALKALOIDS IN TALL FESCUE 173

RESULTS AND DISCUSSION

Cross-reactivityHybridoma clone EN9F10 was found to recognize a broad spectrum of clavine,ergopeptine and lysergic acid derivatives (Table 1, Figure 1). As expected, theantibody showed the greatest affinity for the target EN, as indicated by 50% inhibitionvalues in the CI-ELISA. EN diluted in PBSTM showed a 50% inhibition of absorbanceat a concentration of 464 pg ml"1. With hapten-carrier conjugates, the specificity ofan antibody is directed toward the portion of the hapten most distal to the carrier; bythe method used for these conjugations, the most distal portion of the hapten is theindole end of the ergoline moiety. Our cross-reactivity studies indicate that all fourrings of the ergoline moiety must be intact for recognition to take place. Tryptophan,having only the indole portion of the molecule, and rugulovasine, having a five-membered 'D' ring, are not recognized by the antibody. Additional groups at C-8,including the cyclol peptides of the ergopeptine alkaloids, and shifts in the doublebond at the 8,9 and 9,10 positions of the 'D' ring do not appear to affect therecognition process. In the case of A"9-10 derivatives, where isomerism is possible, theantibody apparently binds to either isomer (ergosine/ergosinine, Table 1).

Analysis of Tall FescueThe CI-ELISA was easily able to detect ergot alkaloids on tall fescue forage sampleswhen as few as 3% of the plants (Table 2) or 4% of seeds (Table 3) were infected withendophyte. This lower limit of detection corresponded to an ergovaline level of 88 ngg"1 and 54 ng g~', respectively, as determined by HPLC. In these and other samples,no false positive ELISA determinations were encountered. Analysis of single freshculms of (Figure 2) was similarly able to detect ergot alkaloids related to endophytepresence, while none was detected in the absence of endophyte. The range ofabsorbance values for non-infected culms did not overlap any values for infectedculms. Samples with extremely low levels of target alkaloids were clearly distinguishedvisually from alkaloid-free samples.

Correlation with Ergovaline ContentAlthough clavine alkaloids as well as ergopeptine and other LA derivatives are presentin endophyte infected tall fescue (Porter et al, 1979; Yates et al, 1985), ergovaline hasstimulated the most interest as a possible cause of fescue toxicosis. Owing to the cross-reactivity of our MAb, most of these compounds would be detected by this assay,however. The comparison of ELISA with HPLC analysis of ergovaline (Tables 2 and3) clearly shows that the results of the ELISA correlate with the presence or absence ofergovaline and that the ELISA detected any of this substance that the HPLC wascapable of detecting. Lyons et al. (1986) demonstrated that 84%-97% of the totalergopeptine content of infected fescue was ergovaline. The lowest ergovaline contentof any sample in the present study was 54 ng g"1 (Table 3). The ELISA is so sensitiveto the presence of ergovaline that most of the samples analyzed (infestation levelsabove 6%) have ergovaline levels that far exceed the 50% inhibition values of theassay. For samples that fall into the high range of ergovaline content, a semi-quantitative assay could be conducted by diluting sample extracts prior to ELISA. Forsamples in the low range of ergovaline content, the dramatic difference between 0%and the 3%-4% infestation levels suggest that the assay would be sensitive to muchlower concentrations of alkaloids without giving false positive results.

Practical Uses of the AssayWe now use the assay in our laboratory to screen samples for A. coenophialuminfestation and concomitant alkaloid production, where qualitative data is sufficient.

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174 R. A. SHELBY & V. C. KELLEY

TABLE 2. Detection of ergot alkaloids in tall fescue plantsinfested with endophyte (A. coenophialium) by CI-ELISA and HPLC

Percentageendophyteinfestation"

0000036

1720204757909393

ELISAabsorbance

490 nm'

1-3211-1831-1201-0101-0800-6770-4430-1450-3270-2910-2080-3380-2780-2600-241

SDe

00800073002100750-1310084002600070032001700150046002000160031

HPLCergovaline(ng g~"0

00000

8876

267202373693783806

18181866

"Determined by microsopy.'Mean of four ELISA wells.'Standard deviation of absorbance values.''Quantitative determination of ergovaline by the method of

Rottinghaus(1991).

TABLE 3. Detection of ergot alkaloids in tall fescue seedinfested with endophyte (A. coenophialum) by CI-ELISA and HPLC

Percentageendophyteinfestation"

00004

161830769296

100

ELISAabsorbance

490 nm»

1-3341-2851-1961-0470-1560-1570-2410-2120-3720-2830-2900-185

SD<

00030009001700630019001301070-0190017003400240018

HPLCergovaline(ng g-"0

0000

54227303

13333864531252688395

"Determined by microsopy.'Mean of four ELISA wells.'Standard deviation of absorbance values.'Quantitative determination of ergovaline by the method of

Rottinghaus(1991).

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DETECTION OF ERGOT ALKALOIDS IN TALL FESCUE

R

175

Compound R RingD

ErgonovmeAgroclavineElymoclavineErgosineErgovalineSetoclavineErgotamineFestuclavine

CONHC (CH3) CH2OHCH3CH2OHCyclol Ala-Pro-LeuCyclol Ala-Pro-Vala-OH,jff-CH3Cyclol Ala-Pro-Phe/?-CH3

A9 '10

A8-'A»'»A'-10

A*'10

A'.ioA'.'"A»*

FIG. 1. Structures of compounds recognized by monoclonal antibody EN9F10.

1.4

1.2

1 1

?n, 0.8u

S 0.6

i< 0.4

0.2

nNON-INFECTEDPLANTS

INFECTEDPLANTS

FIG. 2. CI-ELISA reactions of 10 fresh plants infected with the tall fescue endophyte (A. coenophia-lum) and 10 healthy plants. Vertical bars represent minimum and maximum values.

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1 7 6 R. A. SHELBY & V. C. KELLEY

For example, an initial screen of seed lots by CI-ELISA will invariably reveal thosewith only the slightest level of infestation. Any scored as positive by ELISA can befurther tested by the staining procedure (Shelby & Dalrymple, 1987) for endophytelevels, or by HPLC (Rottinghaus et ai, 1991) for quantitative determination ofergovaline levels. This immunoassay offers several advantages over HPLC for ergotalkaloid determination.

(i) Much smaller samples can be processed. Single plants and individual coloniesof fungi from agar cultures are suitable samples for this method. We haveanalyzed samples as small as 0-5 g with no problems, and samples as small as0-1 g would be sufficient.

(ii) The assay detects alkaloids (such as the clavines) which do not fluorescence,and therefore are not detectable by fluorescence HPLC.

(iii) Elapsed time for analyzing 96 samples is only about 2 h, neglecting samplegrinding. This same sample volume would require eight working days by HPLCin our laboratory.

(iv) The cost of analysis is much less.(v) The test does not require a laboratory environment, and, due to the qualitative

nature of the results, a positive/negative result can be determined visuallywithout a spectrophotometer; the most sophisticated piece of equipment re-quired is a pipette.

(vi) Exposure to hazardous chemicals such as chloroform and acetonitrile areminimized. The OPD used as the chromogen in our assay is a potentialcarcinogen, but other less toxic peroxidase substrates are available.

Other Potential UsesThe cross-reactivity of the antibody and sensitivity of the assay make possible anumber of potential research applications. Bound to a solid phase, the antibody couldbe used to selectively remove and concentrate alkaloids from culture filtrates or otheraqueous complex mixtures as a simplified preparation for quantitative HPLC. Theassay could be used to determine half-life and mode of action studies of ergotpharmaceutical compounds. Radioimmunoassays utilizing antibodies have been usedwith some success (Loeffler & Pierce, 1973; Arens & Zenk, 1980), and enzymeimmunoassays should perform in a similar manner. The specificity of this MAb shouldmake it possible to detect decomposition products of alkaloid metabolism as well asintact alkaloids. The assay also has the potential to be used to screen commoditiessuch as wheat, rye and other cereal grains infested with ergot (Claviceps spp.) and thespectrum or ergot alkaloids produced by these fungi. We intend to explore thesepossibilities in future research.

ACKNOWLEDGEMENTS

The authors would like to thank Michelle Mayo, Charles Backus, Paul Free, RogerBridgman and Larry Dalrymple for technical assistance; Robert Hosford and TrumanOlson, North Dakota State U., and Donald Mathre, Montana State U. for ergotsamples; Kimberly Gwinn, U. of Tennessee, for agroclavine standards; Heinz Floss, U.Washington, for ergotamine and clavine alkaloid standards; Gary Marconi, Eli Lilly,for ergosine and ergosinine; and D. Romer and R. Giger, Sandoz, Basel, for ergocor-nine, ergonine, ergoptine, ergosine, ergostine, ergotinine and ergovaline referencestandards.

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DETECTION OF ERGOT ALKALOIDS IN TALL FESCUE 177

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HARLOW, E. & LANE, D. (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory,New York, NY, USA, 726 pp.

LOEFFLER, L. J. & PIERCE, J. (1973) Radioimmunoassay for lysergide (LSD) in illicit drugs andbiological fluids, Journal of Pharmaceutical Sciences, 62, 1817-1820.

LYONS, P. G, PLATTNER, R. D. & BACON, C. W. (1986) Occurrence of peptide and clavine ergotalkaloids in tall fescue grass, Science, 232, 487-489.

PETROSKY, R. J. & POWELL, R. G. (1991) Preparative separation of complex mixtures by high-speedcountercurrent chromatography, in Naturally Occuring Pest Bioregulators (HADLIN, P. A., Ed.),ACS Symposium Series 441, American Chemical Society, Washington, DC, pp. 426-434.

PORTER, J. K., BACON, C. W. & ROBBINS, J. D. (1979) Ergosine, ergosinine, and chanoclavine I fromEpichloe typhina, Journal of Agricultural and Food Chemistry, 27, 595-598.

REDDICK, B. B., GWINN, K. D., SAVARY, B. J. & COLLINS-SHEPARD, M. M. (1991) Development of animmunoassay for detection of ergoline alkaloids in tall fescue, Tennessee Farm and Home Science,in press.

ROTTINGHAUS, G. E., GARNER, G. B., CORNELL, C. N. & ELLIS, J. L. (1991) HPLC method forquantitating ergovaline in endophyte-infested fescue: seasonal variation of ergovaline levels instems with leaf sheaths, leaf blades, and seed heads, Journal of Agricultural and Food Chemistry,39, 112-115.

SHELBY, R. A. & DALRYMPLE, L. W. (1987) Incidence and distribution of the tall fescue endophyte inthe United States, Plant Disease, 71, 783-786.

STAROS, J. V., WRIGHT, R. W. & SWINGLE, D. M. (1986) Enhancement by N-hydroxysulfosuccinimideof water-soluble carbodiimide-mediated coupling reactions, Analytical Biochemistry, 156,220-222.

YATES, S. G., PLATTNER, R. D. & GARNER, G. B. (1985) Detection of ergopeptine alkaloids inendophyte infected, toxic Ky-31 tall fescue by mass spectrometry/mass spectrometry, Journal ofAgricultural and Food Chemistry, 33, 719-722.

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