Toriran, Vol. I8, pp. 702-7üt.9Pergamon Preis Ltd . 1980 . Printed in Great Britain .

DETECTIONANDQUANTITATION OFAMANITIN USING ANRNA-POLYMERASE COMPETITION BINDING ASSAY

ARNOLD BROWN andGEORGEM. GARRITY

From the Department of Microbiology, Graduate School of Public Health andDepartment of Medicine, University ofPittsburgh School ofMedicine and Veterans

Administration Medical Center, Pittsburgh, PA 15240, U.S.A .

(Acceptedforpublication 4 March 19lß)

IX}S l-II I01/80/1101-0702f02.0pp

ACCIDENTAL intoxication resulting from the ingestion ofAmanitaphalloides andA . vernamaybecome more common as the ranks of less experienced, but adventurous mushroom-pickers expand . Until a successful radioimmunoassay was developed, no direct methodwas available for the quantitation of amatoxins in biological materials (FLUME et al . , 1975 ;FAULSTICH et al., 1975). The radioimmunoassay developed by FLUME et al. (1975) usedantibody prepared in rats immunized with an amanitin-albumin conjugate, whileFAULSTICH et al . (1975) used an amanitin-albumin-polylysine conjugate as an im-munogen in rabbits . Both groups used O-methyl-[3H]-demethylry-amanitin (O-Me3H-DGA) as the radioactive tracer . Problems encountered by these workers were caused bythe residual toxicity of the amanitin conjugates, leading to animal mortality and poorimmunogenicity, resulting in low antibody levels .

In this paper, we describe the adaptation of a competition binding assay to detect andquantitate amanitins. Sensitivity is comparable to that reported for the radioimmuno-assay. Since it uses eukaryotic RNA-polymerase II (E.C.2 .7 .7 .6, nucleoside triphosphate :RNA nucleotidyl transferase, wheatgerm), specificity is at least equal to that of theradioimmunoassay . One major advantage of this assay is that it obviates the need toprepare antibodies to amanitin .

MATERIALS AND METHODS

Synthesis oja radioactive amanitin derivativeO-Methyl-["H}demethyl~y-amanitin was synthesized from precursor a-amanitin by methylation and oxida-

tion to form O-methyl-aldoamanitin . This product was then reduced with sodium boro-['H}hydride to form thefinal product. The details of the synthesis and purification have been described (GARRrrvand BROwN,1978 ;WIELANDand FAHRMETER, I97O) .

Amanitin competition binding assayBinding assays were performed as previ0u51y described (C17('HET-MEILHAC and CAMBDN, 1974 ; GARITY and

BRowN . 1978) . Each reaction mixturecontained500pd ofbinding buffer [80 mMTris^-LiCI pH 7-9 at 5°C, 0-1 mMEDTA, 0-1 mM dithiothreitol, 150 mM (NH,)ZSOa , 0-2 p,g/ml bovine serum albumin, 0-4 mg/ml rabbitJy-globulin and 30°l0 (v/v) glycerol] with8pg enzyme protein, 9 ng O-Me{eH}DGA (ca . 12,000 counts/min, specificactivity 2-0 Ci/mmole) and varying amounts of unlabeled tz-amanitin (50 pg-5 mg). Samples were incubatedoverntght at 4°C at which time an equal volume of saturated (NH,)=SO, was added . Samples were incubated foran additional hour at 4°C and then centrifuged at 29,000 g for 20 min at 4°C. The pellet, containing boundamanitin, was resuspended in 1 ml ofbinding buffer, an equal volume of saturated (NH,)zSO, was added and themixture was incubated at 4°C for 30 min . Following centrifugation, the pellet was reprecipitated two additional

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times. The final pellet was dissolved in 200 N,1 H=O, digested overnight in NCS (NuclearChicago Solubilizer) at45°C, and munted in an non-ageuous based scintillant . a-Amanitin was obtained from Boehringer MannheimBiochemicals (Indianapolis, IN) and wheatgerm RNA polymerise II was purchased from Miles ResearchLaboratories (Elkhart, IN).

The results of the competition binding assay are displayed in Ftg. 1 in the form of a logit-bgplot (RODBARD et al . , 1969). Eight samples were tested at each dilution of a-amanitin andmean values have been plotted. There was a proportional decrease in the amount ofO-Me='H-DGA bound as the concentration of a-amanitin was increased (rz =09840) . Noexperimental values fell outside the 95% confidence limits over the range of 0~5-500 ng .There was, however, a considerable degree of variability when concentrations exceededthese limits . In light ofthe tight, specific binding of toxic amanitins to RNA polymerise II,it is unlikely that these limits in sensitivity are attributable to this component of the assay.The limiting factor in our assay is the specific activity ofthe labeled tracer (2~0 Ci/mmole).Since the specific activity of the O-MesH-DGA used in radioimmunoassays reported byFLUME et al . (1975) and FAULSTICH et al. (1975) was comparable, the same degree ofvariability at low concentrations would be expected .

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RESULTSANDDISCUSSION

Ra I. Ct~tmavetrmnvc.~+YUnlabeled a-amaniti~ in varying rnncentrations (50 pg-5 mg) was reacted with wheatgermRNApolymerise II(8 Wg) in the presenceof9ngcompeting O-Me-~H-DGA. Eight replicate samplesweretested ateach dilutionandmean values of the percentage of trace binding were plotted as a logit-log transformation . Horizontal barsrepresent 95% confidence limits ; rt = 09840.

The major advantage of the competition assay is the specificity of the amanitin-RNApolymerise II interaction . Since the assay iscarriedout in the presence ofa5-fold excess ofRNA polymerise II, it should be relatively free of interference by exogenous bindingproteins . Furthermore, the assay should not be affected by the presence of phalloidinssince these closely related toxins do not bind to RNApolymerise II .Because of the low dissociation rate constant at 0°C for eukaryotic RNA polymerise II

bound amanitin (CoCHE'r-MEILHAC andCHAMBON, 1974), the assay as described wouldonly detect free amanitin . By preincubation at 37°C for 30 min (4~/z half-lives) (Co(~r-

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MEII.HAC and CHAMBON, 1974), before the 18 hr incubation at 4°C, bound amanitinshould equilibrate with the radioactively labeled pool. This modification should allowdetection and quantitation of total (free and bound) amanitin .

Acknowladgantnts-This work was supported by the Veterans Administration Research Service . The excellentsecretarial assistance ofMs . DoNtvw CI~twI~F-Its is appreciated .

REFERENCES

COCHETMEILHAC, M. and C ewt~ISON, P . (1974) Animal DNA-dependent RNA polymerases. 1 l . Mechanism ofthe inhibition of RNA polymerases by amatoxins . biochim . biophys. Acta 353, 160.FnU[.srICtLH ., TRISCHMAN,H . and ZoeeLEV,S . (1975) A radioimmunoassay foramanitin . FEBS Lett. 56, 312 .RUME, L., Busl . C ., GMPADELLI-RU~,G . and FhwNCr scrII,C . (1975) Productionofantibodies to amanitins asthe basis for their radioimmunoassay. Expcritntia 31, 1233 .Gwttttrl'Y.G . and BROwrt,A . (1978) Competition binding assay using O-methyl{aH}demethylsy-amanitin forstudy of RNA polymerise B . Pros. Soc. cxp. Biol. Med . 199, 98.RODBARD, D., BRInsotv, W. and RAYfaRD, P. (1969) Rapid calculation of radioimmunoassay results. ! . clin .Lab. Med. 74, 770.WIELAND,TH.and FAHRMEIER,A . (1970) Oxydation and reducktion an dery~8 dihodroxy-isoleucin-Seitenkettedes O-methyl-a-amanitins, methyl-aldoamanitin, einungiftges abbauprodukt . lustus Litbigs Ann(n Chem . 736,95 .