Table Of ContentIMPACTOFA VALIDATED, COST EFFECTIVESCREEN FOR CIGUATERIC FISH
RICHARD LEWIS
J.
Lewis, RJ. 1994 08 01: Impact of a validated, cost effective screen for ciguatcric fish.
Memoirsofthe QueenslandMuseum34(3):549-553. Brisbane. ISSN0079-8835.
Cigiutoxinscontaminatingciguateric fishmaybedetectedbyarangeofinvivo(e.g.mouse-
cat,mosquitoorchicken),invitro(ELISA.atria)andchemicalassays.Currentresearchseeks
a selective screen todetect low levels ofciguatoxin-1 (0.05-5.0ppb) in fish flesh or in an
easy-to-prepareextract. Thisreviewsummarisesrequirementsforavalidated,costeffective
screenforciguatericfish.Implementationofsuchascreenwillreduceadversehealtheffects.
Anattendantbenefit will be theimproved marketabilityofreeffish.
RichardJ.J^ewis.SouthernFisheriesCentre, QueenslandDepartmentofPrimaryInduslries,
POBox76, DeceptionBay. Queensland4508; 22November.1993.
One important goal of present-day ciguatera alsoundeTdevelopment and this approachholds
research is the development of a cost-effective muchpromiseforthedetectionofciguatericfish.
screen for the toxins contaminating ciguateric These assays remain to be validated for their
fish (Lewis.1993). Therange oftoxins involved ability lo selectively detect ciguatoxins in crude
in ciguatera hasbeenthe subjectofsomedebate. extractsoffish Wevalidatedthemouseassayfor
fIte.agp.pgeaarrnsbtiheantooxnilnyst)hearceigiunavtoolxviends(aMnudraantaaloelguael.s, c&igSuealltionx,i1n99i3n)u.pThtois2a0smsgayowfaesthoenrlyexatbrlaectto(dIext:ewic*t
1990; Legrand et al.,1992; Lewis et a!.,1991) CTX-1 at>0.5ppb in fish flesh. Importantly, this
These toxins are closely related in structure and study found that 63± 1496 of spiked ciguatoxin
all activate voltage-dependent sodium channels. wasrecovered usingastandardextractionproce-
Of these, CTX-1 typically contributes -90% of dure, thereby establishing its suitability for con-
the toxicity ofciguateric carnivorous fish in the firming whether a fish sample was toxic or not.
Pacific (Legrand et a!.,1992; Lewis & Sellin, The cost ofsuch an assay, as well as its insuffi-
3992) and should be considered the primary tar- cient sensitivity and ethical considerations.
get of a screen for ciguateric fish. Other preclude the use ofthe mouse assay for routine
dguatoxin analogues and toxins such as okadaic seafood monitoring programmes. This paper
acid, maitotoxins or Trichodesmium toxins are summarisesrequirementsforavalidated,cost-ef-
Likely to play a minor role in human illness fective screen for ciguateric fish and discusses
However,theselowerpotencytoxinscouldinter- someofthe impactsofsuchatestonfisheriesand
fere withtheresponseofascreen.Thechallenge fisheriesproducts.
for researchers is to develop a method that can
rapidly and selectively screen CTX-1 which is FEATURESOF A USEFUL SCREEN
presentbetween0.1 and5ppb in the flesh offish
thatcauseciguatera(Lewis,1992).Usinga2-fold A useful screen for delecting ciguateric fi^h
risk factor to ensure public health is protected needstopossessthefollowingfeatures. 1, simple
necessitatesthatthe screenbe capableofreliably implementation; 2, ready availability and long
detecting CTX-1 in fish flesh atO.OSppb (50ppi) shelflife; 3, toxinselectivity proportional to the
andabove. human oral potency ofthe ciguatoxin analogues
A number of assays have been used to detect liehighestaffinity forCTX-1); 4, itshould yield
ciguatoxin in fish. These include a range of in a linear response over the range of toxin con-
vivo assays (e.g. mouse, cat chicken, mosquito; centrations encountered in ciguateric fish; 5, ac-
Banneretal.,1960; Kimuraetta)..1982; Lewis & ceptable accuracy (±20%) atthe level of0.05ppb
Endean,1984; Bagnisetal.,1985; Vemouxetal.. CTX-1 that is independent of the fish tested: ft.
1985), a number ofin vitro assays utilising anti- high recovery ofCTX-1 (>60%) during extrac-
bodies (Hokama.1991) or isolated tissues tion and clean-up (>30% in exceptional u-
(KimuraetaL!982)andchemicalassays involv- cumstances); and 7, total cost ofscreen must be
ing derivatisation and HPLC separation with acceptable totheconsumer.
fluorescence detection (Legrand et al.,1992; Prototype stick tests (Hokanria,1985; Hokama
Dickey et al.,1992). Biosensor-type assays are el a).,1985; Hokama 199l) have many of the
(
*w» MEMOIRSOFTHEQUEENSLAND MUSBUM
*bove features, but this or modified versions of give rise to false positive results*. Tb-date die
the test are still not readily available Published CToss-reactivuy of the antibodies in use for the
results of screening of fish suggest ("but do not variousciguatoxinanalogueshasnotbeenestab-
prove) that these tests have sufficient sensitivity lished.
n.c, few false positive results reported). How- Anacceptablecost forciguaterascreening has
ever,theapparenthighfrequencyoffalsepositive nolbeendetermined.Thiswill relatetotheadded
results (i.e. non-toxic fishrejected) suggeststhat value screened fish will attract in the market-
theantibodyemployedmayhavearelatively low place. An add-on cost of less than 10% of the
specificity forCTX-1 value of the product may be reasonable. The
A screen that utilises high affinity binding of 'cost* of a screening programme should incor-
ciguatoxintoa cheapprotein substrate(e.g. IgO) poratean estimateofthecost ofdiscarding non-
andcouplesthisinteractiontoa simplymeasured toxic fish as a result of false positive results It
response(e.g. acolourchange) has a high likeli- may be possible to reduce the cost ofscreening
hood of achieving an adequate compromise be- by pooling fish samples priorto screening. Such
tween accuracy and cost. Such antibody-based anapproachrequiresahighlysensitivescreenbut
screens have the potenMtial to detect ievels of could work where ciguatera is a low level prob-
analytc as low as IO"i2 in food (Gazzaz et aL lem (e.g. Australia). This approach could not
1992). Howrvrr, matrix effects associated with work where non-toxic fish have levels of
thetypeofsamplescreenedcanoftendramatical- ciguatoxinwithinanorderofmagnitudeofleveK
ly reduce assay sensitivity. The extent of sueh thatcausehumanpoisoning.
matrix effects can also vary with the solubility A blind screening of ciguatenc fish ftoflfl
characteristics of the analyte. For solid food Australia (specimens confirmed ciguatenc by
matrices, enzyme linked immunosorbentassays human and mice assays! with a prototype ofthe
(ELISA) detected okadaic acid in shellfish of Ciguatect™ test kit (November,1991) revealed
10-300ppb (DSP-ehcck, Ubc Industries. Ltd, that therewere few. if any. false positive results
Tokyo) and detected aflatoxins above lOp-pb However,merewasastrongpossibilitymatsome
(Domer & Cnle,1989; Trucfcsess et al.,1989) or ciguatoxic samples went undetected by the test
more recently above 1 ppb in a range of solid In fact five of six ofthe ciguateric fish samples
foods (Agri-Screen test for aflatoxins, Neogen are likely to have given a false negative result
Corporation, Michigan). The challenge is to Furthertestingofthese fish usinglatermodifica-
develop a rapid screen for ciguatoxins that has tionsofthe Ciguatect™ testare notreported and
onetotwoordersofmagnitudegreatersensitivity nosatisfactoryexplanationhasbeengivenforthe
than presently availableELISAassays. A variety conflicting results obtained. These results were
ofapproaches may be used to improve the sen- obtained at a time when the test was being con-
sitivity ofantibody-based assays: (i) production sidered for commercial release. Since this time
ofhigheraffinityantibodies(ii)optimisingassay modifications to the test have been made but a
conditions in which the antibody is used (ill] final format for the Ciguatect™ test remains
amplifying the assay signal (not always accom- elusive.
paniedbyimprovedsignal tonoise)(iv)optimis
ing sample extraction and clean-up (can add a TYPESOFANTIBODYASSAYS
significant cost). The potential of the latter ap- AVAILABLE
proach is indicated for ciguatoxin which can be
concentrated fromthelevelsin fleshof0.1-5ppb Several approaches are available for incor-
tolevelsof-3O-5.0O0ppbinacrude lipidextract porating antibodies (or similar proteins) into an
withatwo-step clean-upprocedure thathas63% assay format for detecting haptens such as
efficiency (Lewis& Sellin.1993). ciguatoxin (Fig.l). In all cases a label, be n a
Toxins responsible forciguatera anse through radioisotope, an enzyme or a luminescent or
the biotransformation of a number of gambier- fluorescent probe, is used to detect the targeted
toxins produced by Gambierdiscus toxicity compound. Each approach has strengths and
(Murataetal ,1990; HolmesetaL1991;Legrand weaknesses hut all require a high affinity an-
et al.,1992). Soa rangeoflow potency formsof tibody that is selective and specific for the tar-
the ciguatoxins and gambiertoxins (including geted hapten. The first assay considered is the
CTX-2 and CTX-3) could accumulate in fish. indirecthapten assay which requires thatthetar-
These low potency forms may be detected with geted hapten (eg. ciguatoxin) is first non-selec-
antibodies raised to CTX-1 and ifso they could tivelyimmobilisedtoa solidsupport(alongwith
SCREENFORCIGUATERICFISH 551
INDIRECT HAPTEN ASSAY extraction step. Tests developed for ciguatoxins
by Hokama (1991) and morerecentlyby Hawaii
Chemtect (Pasadena) have used this approach in
the development ofa screen forciguateric fish.
The second approach is to develop a sandwich
assay. This requires development of two an-
£><>(—) tibodies which can mutually bind the targeted
hapten.Thisassaycanbemoreselectivethanthe
direct assay although binding reactions in two-
non-selective adsorption o( haptens to the solid phase site immunoassays are complex and not easily
predicted (Boscato et al.,1989). This assay is
limitedbythedifficultiesassociated withobtain-
SANDWICH ASSAY ing two antibodies which don't interfere with
each others binding to a small hapten such as
ciguatoxin.
The third approach is the direct competitive
assay which requires that a second hapten is
available that competes with the targeted hapten
for binding to an antibody. With this approach
eitherthe hapten or the antibody can be fixed to
the solid phase. Unlike the first two assays, the
unlabeled antibodies HgGI selctively fixed to the solid phase responseis inversely proportional to thetargeted
hapten, but such a response is likely to be more
specific for the targeted hapten than the former
assays. Thedirectcompetitive assay likely holds
DIRECT COMPETITIVE ASSAY
mostpromise foran accurate screen but requires
a competing hapten to be found. While it is pos-
sible that the sandwich and competitive assays
couldbedevelopedforthedirectsamplingoffish
flesh, it is likely is that extraction and clean-up
steps will be required. The solubility require-
ments of the hapten and stability of antibody in
solvent need to be considered if the assay is
unlabeled antibodies HgGI selctively fixed to ihe solid phase designed to detect solubilised hapten.
FIG.l. Antibody assays available for the detection of
haptens suchasciguatoxin(CTX). Eitherthe IgGor SCREENVALIDATION
hapten are labelled (L) with probe to indicate the
presence ofthetargeted hapten. Other shapes repre- A number of criteria need to be met before a
sent the range ofcompounds present along with the screen forciguateric fish can be considered use-
targetedhapten.TheIgGcouldbereplacedwithother ful, including: 1, the screen must detect all fish
proteins possessing a high affinity for the targeted samples confirmed ciguateric following human
hapten (eg the sodium channel could be used for
detectingciguatoxin).Thelargeboxineachdiagram consumption; 2, the screen musthave an accept-
surrounds the interaction responding tothepresence ably low rate offalse positives (i.e. a false posi-
oftargetedhapten,whileinteractionsbeyondthisbox tive rate within an order of magnitude of the
resultinareduced(falsenegative)orenhanced(false reported ciguatera incidence for the species
positive) assay response that is unrelated to the tested) and the cost offalse positive results must
presenceofthetargetedhapten. beincluded inthecostofscreening; 3, thescreen
must detect spiked CTX-1 in crude fish extracts
numerous othercontaminants) priorto its detec- at levels occurring naturally and should detect
tion with a labelled antibody specific forthe part CTX-1 spiked in a fish flesh homogenate; 4, the
ofthe hapten left exposed following binding. In screen should produce appropriately accurate
practice, it is often not possible to obtain an results for toxic and non-toxic fish both within
antibody that can still 'see' small haptens bound andbetween laboratories. Whereverappropriate,
toasolidphase,butthisapproachcanallowrapid negative controls should be run in a pair-wise
detection ofa hapten without a time-consuming design and results forthese shouldbenegative.
552 MF.MOrRS OPTHEQL^ENSLANDMUSEUM
Absence of readily available tefeience toxins increase if more than one point of testing is re-
of the ciguatoxin class and lack ofa validated quired. It is interesting tospeculateon the fateof
analytical methodtoquantify the level amitypes a screening product thai is shown to have failed
of toxins present in samples of fish hinder at- todelectatoxic fish,especially ifthe fish results
tempts to validate screening tests for ciguateric in apoisoningepisode.
fish. The use of the mouse bioassay to assess Anotherissuetobe considered is what fishare
ciguatoxin levels (Lewis & Sellin in press) is to be screened In some areas it might be ap-
presently the best available alternative to an in propriate toscreen all potentially ciguateric reef
vitro approach. It could prove misleading to use fish, whereas in other areas only the high risk
otherhivivobioassaystovalidatescreening tests species presently marketed may need lo be
atthistime,especiallytheunreliablebrineshrimp screened.Otherclassesoffish thatmay requirea
assay(RJ. Lewisunpuhl. data) winchhas recent- differential approach include (i) fish presently
ly been used by D.L. Park to characterise the banned as a result ofthe threatofciguatera they
toxins presentin fish screened by the S-PIA ver- pose (li) fish destined for export and (lii) large
sionofthe Ciguatect™test(Hungerford,1993). whole fish inoneofthe abovecategories.
Toensurcreliabilityofascreen,anylimitations
of a screen with regard to species, sample LITERATURECITED
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toxic and non-toxu- Speci0ie.Q$ to ensure ay, (he mouse bioassay and the mosquitubtoas-
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