Table Of ContentEVALUATION OFASOLID-PHASE1MMUNOBEADASSAYFOR DETECTION OF
CIGUATERA-RELATEDBIOTOXINS INCARIBBEAN FTNFISH
ROBERTWi DICKEY,H.RAYGRANADEAND FOSTER D. McCLURE
&
Dickey, R.W., Granade, H.R. MeClure, F.D. 1994 08 01: Evaluation ofa solid-phase
immunobcadassayfordetectionofciguatera-relatedbiotoxinsinCaribbeanfinfish.Memoirs
oftheQueenslandMuseum34(3): 481-488. Brisbane. ISSN0079-8835.
TheCiguatect™solid-phaseimmunobcadassayfordetectionofcigualcra-relaicdpolyether
biotoxinsinfinfishwasevaluatedforconsistencywiththemousebioassay Fiftyfinfisnfrom
.
ciguatera-endemic waters ofSt. Thomas,U.S. VirginIslands, and one fish remnant froma
confirmedcaseofciguaterapoisoningweremousebioassayed.The51 specimenswerethen
assayed using the Ciguatect™ assay with 3 different methods of tissue sampling: single
exposure, tripleexposure andsingleexposure to solventextractfrom flesh (REM™: rapid
extractmethod).Qualitativestatistical analysesascertainedfalse positiveandfalsenegative
REM™
rales. Positive matches forthesingle, triple and methods oftissue sampling were
58, 85 and 94%, respectively, and negative matches were 17, 22 and 12%, icspcciivcly.
Corresponding false negative rates were 82, 5™5 and 50%, and false positive rates were 44,
33and33%. PredictiveindicesforCiguatect performanceunderciguatoxincontamina-
tion ratesranging from 5 to 75% project thathigh false negative and false possuve values
mightbeexpectedinmarket situations.
Robert W. Dickey & H. Ray Granade, GulfCoastSeafoodLaboratory, OfficeofSeafood,
Food and Drug Administration, P.O. Box J5H, Dauphin Island, AL 36528: Foster D.
McCiure, Division ofMathematics. Food and Dtuv, Administration, 200 C Street, S W.,
Washington, DC20204; 28March. 1994.
The major impediment to management strat- rotary evaporatorModelRE-111 (BrinkmannIn-
egies fortheciguaterapublic healthhazard is the struments, Inc., Westbury, NY); Meltler Model
difficulty in detecting highly potent ciguatoxms PM2000top-loadingbalanceI'MettlerInstrument
(and roaitotoxins) in seafood matrices. Cigua- Corp.. Hightstown, NJ); Sartorius Model R180D
toxin and maitotoxin structures have been eluc- analytical balance (Sartorius Corp., Bohemia,
idated from Pacific sources (MuraUi £1 a1.v 1989, NY); S.ivant Model SS-2 centrifugal evaporator
1990,1992J993;Lewisetal ,1991; Yokx.yamaet (Savanl Instruments, Inc., Farmingdale, NY),
al.J988; Holmes et ah, 1990). but Caribbean
forms have not been resolved and analytical Reagentsand Materials
methods to detect and quantify them srz not yet Solid-phase immunobead assay (SP1A)
available.Severalpotentialassessor)'toxinshave materialswerepurchasedfromHawaiiChemted
also been characterized (Murakami et aI..1982: International. Inc. (San Diego, CA) on 24 May
Tohgoe et al.,1988; Nagai et al.,1992, Fukui et 1992 (immunobead lot number 051492) and
aLs 1987). Difficultieswith developingciguatera again on 6October 1992 (immunobead lot num-
detection methods are due to lack ofanalytical ber 100592). All solvents were ofreagent grade
standards. An immunochemical assay fordetec- orbetter(J.T. BakerChemical Co.,Fhillipsburg,
tion of ciguatera-related poJvethers was devel- NJ). Silica gel solid-phase extraction columns
oped (Hokama,1985,1990; Hokama et al.,1990, wereobtainedfromVarianAssociates, Inc.(Sun-
1992) and modified into a 'kit* format, jle. CAi. Swiss white mice (Crl:CFW
'Ciguaiect™* (Park et al.,1992). We compare (SW)BR) were obtained from Charles River
this assay with the mouse bioassay, the most Laboratories (Wilmington, MA).
widely recognized method for identification of
ciguatoxic finfish. SpecimenCollection
Potentially ciguatoxic finfish were collected
EXPERIMENTAL 4-13 June.1992 from ciguatcra-endcmic waters
S ofSt. Thomas, U.S. Virgin Islands (McMillan
Apparatus et ai.,1983). Fish weresolicitedfromlocalspons
Eberbach 80Pexplosion-proofblenderlEber- fishermen. Fish were collected by spearfishing.
bach Corp., Ann Arbor, Ml); Brinkmann/Buchi Participantsinanannualfishingtournamentwere
4^ MEMOIRS OFTHEQUEENSLAND MUSEUM
notifiedoftheneedforspecimens,anddonations SPE products by vacuum centrifugation. The
of fish were accepted at the weigh-in station. residueswereredissolvedinknowntotalvolumes
Larger fish of suspect species frum areas noted ofsaline(0.15M NaCl)containing l%Tween-60.
for ciguatera were procured as ihey have the Appropriatealiquots ofthe saline solutions were
highest probability ofbeing ciguatoxic. This, ap- taken such that the dosing of mice was equal to
proach wastakenknowing thata significant per- muscle tissue fresh weightequivalents(MTE)of
centage of finfish are non-ciguatoxic even from 45, 90 or 180g per mouse. Residue weights ad-
highincidenceareas.Specimens wereidentified, ministered to mice did not exceed 20mg, and
weighed, tagged, frozen and air-freighted to the ranged as "low as 318u,g when adjusted to the
FDA Gulf Coast Seafood Laboratory (GCSL) chosen MTE. All bioassay solutions were ad-
?
Dauphin Island, AL. All specimens arrived justed to a total injection volumeof0.5mLprior
frozen with no evidence of thawing or decom- lo bioassay. The 0.5mL solutions were ad-
position. Fishspecimenswerestored at -2CfC. ministeredby intraperitoneal injectionintoSwiss
white mice (Crl:CFW(S\ViBR) weighing 18-
TestSamplePreparation 21g. Control mice were administered saline-
Fifty Caribbean fish were selected, including Tweensolution only. The mouse bioassays were
Sj>hyraetta barracuda (7(Wt), Cararvc latus (20 performed in duplicate forcontrols and for each
>• {2%)*Serioladumen Seri- MTE of the final SPE products. Mice were ob-
via rivuiiatia (2%) and Scomberoworus cavaila served post-injection fora period of48hr. Signs
{2%). From each specimen two muscle tissue oftoxicity were noted and when death occurred,
samples were takenfromtheanterolateral partof limesfrominjectionwererecorded.Mousebioas-
the body immediately behind the head: lOg for wetc conducted according to the principles
useintheSPIAprocedureand450gforextraction provided in tin* Guidefor the Care and Use ../
and mouse bioassay. Samples, also were taken Laboratory Attimalw Institute of Labors
froma filletportion ofbarracuda (specimen VI- Animals Resources, National Research Council.
108: courtesyofDr Norbert M t, Thomas NIH Pub. No. 85-24.
Hospital) that was responsible for ciguatera
poisoning in St. Thomas and from a Cynoscion C]GUATRT™SPIA
arenarius (white trout: specimen 92-07-1; not A 9x10mm white membrane, affixed lo a
included in the tables) obtained from the retail 90x9mm plastic strip is exposedto muscletissue
seafoodmarketofDauphinIsland,AL,asanega- taken from behind the head of the fish. The
tivecontrol. Aportionofthe latterspecimenwas membrane is permitted to air-dry and is then
consumedwithouttoxic effect.The !0g samples immersed in methanol for no more than Isec.
were frozen at -2(fC. The 450g samples were After the membrane is completely dry, it is im-
weighed to the nearest O.lg, and extracted and mersedinananti-ciguatoxin-aniihody-latexbead
partitioned (McMillanet ah, 1983; Yasumotoet suspension (gently mixed prior to use) for 5 or
al.,1984).Thedried products,from the extraction lOtnin.The lattersolutionisblue. Themembrane
and partitioning procedures were dissolved in isthendipped inandoutofa phosphate-buffered
chloroform and appliedto 60mL (lOg) s'dic saline solution 3 times, placed flaton an absorb
solid-phase extraction (SPE) columns that had em towel and gently blotted to remove excess
been preconditioned by washing with 60mL of saline dilution. Adsorptionofciguaterabii'tnxir^
the same solvent.TheSPEcolumnswerewashed to the membrane and subsequent conjugation
with an additional 120mL chloroform and then with the anti-ciguatoxin antibody bound to the
elutedwith 120mL 10*3: methanolinchloroform. surface ofthe blue latex beads should produce a
The eluting solvent mixtures were removed by blue color (cast) in the membrane affixed to the
rotary evaporation, and residues transferred in plastic strip if the fish contains ciguatera
methanol (c.2-3mL)to tared 13x100mm Teflon- biotoxins, Kit positive-control membrane strips
cappedtesttubes. Solventwasremo inby are processed from the point of exposure to an-
vacuumcemrifugatinn and residueweighs were tibody-latex bend solution- Kit negative-control
recorded to the nearest 0.1mg for each final membrane strips arc processed from the™ooint of
product Final SPB products wetc dissolved in exposure to methanol All Ciguatect SWA
methanol (e.l-2mL) andstored at -20**17. materials arc stored at 4^X7.
Instructions for the first set of mate;
Md
Si- BlOASSAYS (teceh ed 24 May 19(92) directed the userto ex-
Methanol was removed fromthe muscletissue pose the membranes to fish tissue only one time
IMMUNOBEADASSAY FOR BIOTOXINS INCARIBBEAN FISH 483
TABLE 1. Mouse bioassaydosing, fish muscle tissue Specimen 45gMTE 45gMTE 90gMTE 90gMTE
deqousievaolfe1nt8s0g(MMTTEE)aadmnidnissutrevirveadltotimmiecse..* indicates (mdgo/skeg) tsiumrevi(vharl) (mdgo/skeg) tsiumrevi(vharl)_
VI-67 51.5 1.51 107.3 0.92
Specimen 45gMTE 45gMTE 90gMTE 90gMTE 51.8 >48 113.8 0.91
dose survival dose survival VI-70 195.3 >48
(mg/kg) time(hr) (mg/kg) time(hr) 210.8 >48
VI-13 46.0 >48 83.9 >48 VI-71 33.3 >48 62.9 2.50
42.6 >48 77.9 >48 34.1 >48 70.2 14.25
VI-17 1018.9 0.20* VI-72 35.9 >48 62.1 >48
856.9 0.36* 31.6 >48 63.7 >48
VI-26 369.0 25.00 Vl-73 41.1 >48 73.1 1.10
337.8 >48 40.2 >48 72.1 4.66
VI-27 258.7 3.60 VI-75 110.6 >48 202.3 >48
236.8 1.80 120.3 >48 218.4 >48
VI-32 301.0 >48 VI-76 44.1 >48 81.8 >48
275.4 >48 39.7 >48 79.0 1.68
VI-33 183.8 0.33 VI-77 52.7 0.66
219.3 0.32
45.4 0.63
VI-34 255.4 1.9 VI-79 84.2 2.13
230.6 13.00 83.3 >48
VI-35 265.2 1.00 VI-81 69.7 >48 131.9 >48
250.5 1.56 61.4 >48 128.5 >48
VI-37 295.7 0.55 VI-85 44.1 2.92 93.8 1.21
365.3 0.63
41.3 5.50 92.8 1.61
VI-38 122.5 0.33 VI-86 54.9 28.00
119.3 0.63
56.4 4.50
VI-48 73.3 >48 121.3 >48 VI-88 236.3 >48
65.3 >48 130.7 2.21 238.7 >48
VI-49 80.3 0.15 VI-90 44.1 15.50
68.6 0.15 40.1 >48
VI-52 17.4 0.66 34.4 >1.00 VI-91 67.0 1.53
15.8 0.45 35.6 >1.00 66.7 >48
VI-53 82.1 2.50 VI-92 27.4 >48 50.8 >48
79.8 6.40 26.2 >48 45.5 >48
VI-54 7.9 >48 15.2 >48 VI-93 70.7 >48 127.2 >48
8.7 >48 14.9 >48 71.5 >48 127.2 >48
VI-55 135.8 >48 218.0 >48 Vl-97 74.5 >48 128.3 >48
124.4 >48 229.2 >48 74.1 >48 130.1 >48
VI-57 56.8 0.50 VI-98 78.0 >48 123.0 >48
53.0 0.45 71.1 >48 129.1 >48
VI-58 63.4 >48 126.0 0.61 VI-99 61.3 0.86 138.8 0.10
75.3 >48 149.4 0.30 61.3 0.88 123.1 0.23
VI-59 123.6 >48 VI-100 54.1 >48
121.8 >48 54.4 >48
VI-60 73.2 >48 133.3 >48 VI-102 46.2 >48
73.6 >48 141.8 >48 44.9 >48
VI-61 131.5 3.0 VI-105 288.1 0.13*
133.0 >48 278.6 0.55*
VI-62 74.9 6.10 VI-106 64.0 >48 120.2 0.15
76.1 >48 62.3 >48 124.6 0.15
VI-63 87.4 >48 157.7 >48 VI-107 54.3 >48 90.9 1.10
90.5 >48 154.5 >48 48.7 >48 90.0 0.73
VI-64 175.6 15.50 VI-108 55.6 >48 146.0 0.83
170.4 >48 55.6 >48 153.7 1.83
VI-65 81.7 >48 162.2 16.00 400.0 0.35*
83.4 >48 146.9 >48 412.6 0.22*
484 MEMOIRS OFTHEQUEENSLANDMUSEUM
Spec- singleexposure tripleexposure RKM™ Mouse Spec- singleexposure tnpleexposure REM™!Mouse
imen Mean SO Mean SD duplic- bioas- imen Mean SD Mean SD duplic- bioas-
ates* say ates* uay
VI-13 t.5 03 0.2 0.4 3,3 N VI-OX) 1.1 0,7 2.5 i 0.1 N
' Vl-17 1.8 0.4 1.8 0.4 0,1 T VI-102 0.5 0.5 2.2 0.4 3.3 N
VI-26 2.3 05 2.3 0.5 5,5 T VI-105 0.8 ! 0.4 2.7 1.7 4,4 T
Vl-27 1.8 0.4 4.0 0.0 5.6f ! VI-106 12 0.7 1.8 0.4 2.2 T
vJ-.-.J 1.8 0.4 3.8 1.2 3J N VT-107 2.0 0.0 l 5 0.5 2.2 T
VI-33 3.2 0.7 3.2 0.4 4,4 i VI-108 0.5 0.5 2.2 0.4 5.5 T
VJ-34 2.5 0.5 3.8 0.4 3 ? 1
Vl-35 2.0 Q 3.0 0.6 5,5 T TABLE 2.Ciguatect™ SPIA mean scores (n=5) and
VI-37 3.7 0.5 2.0 0.0 4,5 T standard deviations (SD) forthree variations ofpro-
Vl-38 0-5 0.5 3.7 0.5 5.5 T cfaeldsuere,negBaotlidvfeacsecovraelsuerseliantdiivceatteo fmaolsueseposhiitoiavsesaoyt
VI-48 0.7 0.5 1.8 0.4 >.3 T results(T=ciguatoxic; N=survival beyond48hr). *
VI-49 1.5 0.5 0.7 0.5 2.2 T indicatesdatasetprovidedby DrD.L. Park, Univ. of
Vl-52 0.5 0.5 3.7 0.9 2,2 T Arizona, t/indicates apparent inconsistency with
regardto immunoassay scoring scale.
Vl-53 1.8 0.4 2 7 1 ' 3.3 T
Vl-54 18 O.J 2 5 0.5 3.3 N single exposure) and to allow the membrane to
•
VI-55 i3 0.5 1.2 0.4 N dry before proceeding. Weakcolor development
| VI-57 1 o o.o 2 7 ! 2 5,5 T prompted themanufacturerto modify the proce-
VI-58 0.8 0.4 33 l.i 5.6t 1 dure for the second set of materials (received 6
Vl-59 23 0.4 2.7 l.t 5,6t N October 1992). The latter procedure instructed
theusertoexposethemembranesbycontactwith
vi r>o 2.0 0.0 3.8 i.3 5.5 N fish tissue three times (triple exposure) witil
T
V1-61 1.5 0.5 1.8 0.4 2 2 drying periods following each exposure. Conse-
VJ-62 I.I) O.ii 2.3 1.1.5 1,1 T quently. Ciguatect™ immunoassays were per-
Vl-63 2.5 0.5 2.7 U 7 U N formed twice on each lOg test sample. The
VI-64 1.8 O.J 3.0 0.8 4,4 T instructions for immunoassay setup, fish assay
VI-65 0.2 0-4 03 0.5 4.5 T anduseofpositiveand negativekitcontrolswere
VVIl--6770 31..70 067 23..30 00,.56 43.45 NT tdfeeoslvtlesoltworepidpesdpr(peprcriioosvreiltydo.edfPiowsshiittthiivsSesuPaeInAdasnsmeaagytsae.triiTvahelsec)oSnwtPerIroAel
VI-7I n.7 ixs 1.3 as 5,5 r color developments for controls and fish tissues
Vi-72 1.8 0_4 1.3 0.7 4,5 N were read and interpreted independently by 6
:
VI-73 0.8 04 0.0 5,5 ! analysts.Participantswereaskedtoassignavalue
VMS 13 05 I .: 04 4,4 N of (no color) to 5 (strong color) to color dev-
Vl-76 in 00 1.0 ao 2,3 T elopmentofeach teststrip by comparison with a
Vt-77 1,7 0.7 i 2 04 3.3 T stetsalndstarridpssseruipepslioefdpboysitthievekiatnmdannuefgaacttiuvreerc.ontrol
VI-79 2.0 1.3 0.5 5,5 7 The lOg test samples that were used for
VI-8I 0.8 0.4 1.0 QUO 4,5 N Ciguatect™ SPIA evaluation at the FDA GCSL
Vl-85 1.7 0.7 2.0 0.6 5r5 T were frozen and delivered 'blind' (i.e., cacti
Vl-Xfi 3.5 i :i 2.5 0.8 5,5 T portion was identified only by code number) to
VI-88 33 0.5 2.D 0.0 4,4 N thelaboratoryofthekitmanufacturerforarepeti-
Vl-90 0.3 Ci 5 [>. 0.4 3.3 7 tion ofthe SPIA procedures.
V1-91 tl 3 LI 5 I 5 1.6 4,4 1 StatisticalAnalysis
Vl-92 i) ; ,i.5 0.7 u 5 43 N The Ciguatect™ SPIA method was assessed
M
Vl-93 i.l 0.4 !.5 5 N t using procedures for evaluating screening tests
«
VI-97 2.0 0.0 0u.0 M ! N (Fleiss,1981i. Method performance is described
VJ-9S J.3 0.7 2» N through four interrelated rates: sensitivity.
.
VI-99 1 1* 0.4 0.8 a i , ^33 1 sspeencsiiftiicviittyy,rfaalles(ePp+o)siitsitvhee,parnodpofarltsieonneogaftciovrer.ecTthley
,
1MMUN0BEAUASSAY FDR BIOTOXINS IN CARIBBEAN FISH 4*5
TABLE 3Ciguatect™ SPIA performance rates rela- sivejumping; straubtail;convulsions;anddeath.
tive to mouse bioassay results. * indicates data <ei Not all mice displayed each of these signs of
providedby DrD.L. Park, University ofArizona. toxicity. Some characteristic signs of toxicity
Performance Performancerale REM™ (w1e5r/e33n:ot4o5bs%e)rveIdnin5m0i%ceotfhastheoxrptirseudrvwiivtahlisn tIhher
criterion eSxipnogsluer-e eTxrpiopstuer-e immuno- dosage was icduced to 45j MTE (lowest dose
immunoassay immunoassay assay* level administered; and longer survival timesor
ENSnTVTTY 19/33(58<fe) 28/33(8.V*> 31/33(94%) survivals through 48hr were noted with chaisac
igilS Of toxicity. The mouse bioassiiy 01
SPECIFICITY 3/18(17*1 4/18(22%! 2/1702%) specimen VI-108 resulted in survival beyond
48hrat45g MTE, survivaltimesof0.83 and 1.83
FALSE 14/17(R2^| 5/9(55% 2/4(50%) ai 90g MTE, and survival timesof0.35 and 0,22
NEGATIVE I at I80g MTE. Specimen 92-07-1 resulted in sur-
FALSE 15/34(44%) 14/42(33%) 15/46(33" vival beyond48hrat 180gMTE withoutanysign
POSITIVE of toxicity. Fish specimen* from which cor-
responding SPE productsproduced mousedeath
classified known' positive ten samples. Spec- with characteristic signsoftoxicity at 90g MTE
ificity rate i.P_) is the proportion of correctly were considered ciguatoxic
classified "known7 negative test samples, False
positive rate (PF*) is the proportion of positive CWUATECT™SPIA
classified test samples that are misclas>ified SPIA eating*ofthe fishspecimens werescaled
'known' negatives. False negative rate {PR-) is from to5 bycomparison ofcolordevelopment
the proportion ofnegativeclassified testsamples with a standaid series of pre-deveioped mem-
that are misclassified 'known' positives. A branes supplied by the manufacturer. The
'known' positiveor'known' negativetestsample manufacturerspecifiedthat ratingsof or 1 were
isatestsamplethathasbeenclassifiedaspositive to be considered non-ciguatoxic and ratings
or negative by the reference method (i.e., mouse above 1 through 5 ciguatoxic at progressively
bioassay). False positiveand false negative rates, greaterlevels.Themeansandstandarddeviations
as defined by Fleiss. are predictive rates. These of ratings for SPIA responses were recorded
wereusedtoassesstheCiguatcet1MSPTAmethod (Tabic2). However, forcomparison with mouse
when used on populations offish with different bioassays the specimens were scored as cithci
proportionsofciguatoxicspecimens. positive (ciguatoxic) or negative tnon-cigua-
toxie) by SPIA. Results from single exposureof
RESULTS ANDDISCUSSION .trip membraocs to muscle tissues indicated
that 34/51 (67%) of fish specimens were
MouseBioassay ciguatoxic.Basedupontheprecedent (McMillan
Characteristic signs ofciguatoxicily(Hoffman etal.,1983;Yasumotoetal.,1984; Hoffmanel al
el al..l983; Kimuraelal.,1982)anddeath within 1983; Kimura et al.,1982: Vernoux et al.. 1985)
48hr were chosen as the determinants for the that the reference mouse bioassay accurately
presence ofciguatera-related biotoxins. The first reflects ciguatoxicily, results of the 3 sample
three fish SPE products bioassayed were ad- treatmentmethods withtheimmunoassaytechni-
ministered atdosesof ISOgMTE. Survival times que are shown in Table 3. Single-exposure SPIA
were short for the three products (=^0.36hr). All scored the human illness case sample (specimen
subsequent bioassays wereconductedatan MTE VI-108) negative (mean 0.5, SD=0.5) and the
ofeither90or45ginordertorecordthepresence non-toxic white trout specimen positive (mean
0T absence of characteristic signs of cigua- 2 7, SD=0.7). The SPIA results following triple
toxicily. The mouse bioassays resulted in 33751 exposure of test-strip membranes to muscle tis-
(65%)mortalities(Tabic 1).Atotalof79pairsof sues indicated that 42/51 (8J%] of the fish
mice were injected (duplicate bioassay for each specimens were ciguatoxic. Triple-exposun:
MTE) In 10 of ihe 79 pairs 1 mouse expired SPIA scored VI-108(mean 2.2. SD=0.4) and the
within48hr, and theothersurvived beyond4Shr. white trout specimen (mean 4.0, SD=0.5) both
SfgttS Of ciguatoxicily in mice included i positive Mean scoresforthekitinternalcontrols
tivity;pilocrcction; vasodilationinears;cyanosis for single exposure of test strips included 2/2
oftail, feet and muzzle; lacrimauon: salivation; positive matches and 2/2 negative matches.
diarrhea; dyspnea; unsteadygait; tremor/convul- Scoresforkit internalcontrolsfortripleexposure
486 MEMOIRS OFTHEQUEENSLANDMUSEUM
TABLE 4.Predictive indices of Ciguatect™ perfor- products and for possible non-specific toxicity
mance,givenciguatoxinscontaminationratesfrom5 unrelated to ciguatera in the mouse bioassay. A
to75%. PF+=falsepositive; PF. =false negative. repeatsampling of 13 muscle tissues (25%)from
the original 51 fish provided a cross-section of
Con- Predictiveindex
tamin- REM™ tissue that were SPIA positive and negative,
ation Singleexposure Tripleexposure mousebioassay positive and negative, and SPIA
(%) PF+ PF_ PF+ PF_ PF+ PF_ falsepositiveandfalsenegative.Thetissueswere
analyzed for putrescine and cadaverine by the
155 00..89961439 00..13118010 00..98435876 00..01304764 00..89446826 00..80323634 hmiestthaomdinoefSbtyartuhsezfkliueowriocmze&triBconmdet(h1o98d1()AanOdACfo,r
25 0.8128 0.4590 0.7333 0.1852 0.7444 0.1466 1990). Determination ofputrescine was not pos-
35 0.7288 0.5782 0.6300 0.2685 0.6432 0.2172 sible because ofmatrix interference. Cadaverine
45 0.6389 0.6756 0.5284 0.3581 0.5426 0.2965 levelswerebelowthelowestcalibrationstandard
55 0.5422 0.7566 0.4286 0.4545 0.4426 0.3864 of 0.5u-g/g in 10 of the 13 tissues. Three of the
65 0.4380 0.8254 0.3305 0.5587 0.3432 0.4889 tissuescontained 3.7,7.2and8.4u,g/gcadaverine
(Table 5). Cadaverine levels above 6u.g/g in
75 0.3254 0.8842 0.2340 0.6716 0.2444 0.6071
tunafish indicate decomposition. Tolerance
levels for other species of fish (including those
of test strips included 2/2 positive matches, 1/2
n1e.2gaotnive5-mpoaitncthesscalaen)d. F1aflaslesenepgoastiitvivees (wmeeraennootf uHsiesdtaimnintehelepverlessednitdnsottudeyx)ceaerde 0n.o1tmegst%abilnis1h1edo.f
observedamong™the kitcontrol test strips. the 13 tissues, and did not exceed 0.2mg% in the
The Ciguatect SP1A resultsobtained by the tworemainingtissues.Thedefectactionlevelfor
manufacturer's laboratory for the fish test histamine in the United States is 20mg%, and
samples evaluated by FDA GCSL do not cor- 50mg% poses ahuman health hazard. There was
respond to results obtained by GCSL. The no correlation between cadaverine or histamine
manufacturer laboratory First performed extrac- and SPIA ormouse bioassay findings.
tion and partitioning procedures (REM™ :rapid Thirteentissueextractproducts(corresponding
extraction method) on each test sample. The to the specimens analyzed for decomposition
SP1A was then performed on the REMIM products) were evaluated forsodium channel ac-
products (Tables 2, 3). SPIA results obtained by tivity by using a telrazolium-based neuroblas-
followingthe REM modification oftheproce- toma cell bioassay for neurotoxins active on
dure indicated that 46/50 (92%) of the fish sodium channels (Manger et al.,1993). Sodium
specimens were ciguatoxic. VI-108 scored posi- channel potentiating activity is indicative ofthe
tive (duplicate scores of5 and 5). ciguatoxins. The bioassay indicated that sodium
Thesensitivityandspecificitytestperformance channel activity was present in all 13 tissue
rates for each variation of the SPIA procedure products and provided a general ranking of
(Table 3) were used to evaluate how the sodium channel activity for those tested (Table
Ciguatect™SPIA wouldbeexpectedtoperform 5). The activity ranking correlated well with
when used on fish populations with different mousebioassaysurvival times. Specimenextract
proportions of ciguatoxic specimens. Table 4 products shown to be highly toxic by the mouse
presents a summary of the predictive values for bioassay also produced significant reductions in
each SPIA method by population ciguatoxicity cell viability through sodium channel potentiat-
rate.Assumingthatatrueciguatoxinscontamina- ing effects. Inhibitory doses which reduced cell
tloitonofrattreoopfic5al5%fisihs,etnhceoupnretdeircetdivien ainhdyipcoetshgeetniecarl- rviaanbgielidtfyrobmy29t0o%6magftMerTE22pehrr20e0xmpiocsruorleite(rIDc9e0l)l
ated from the present study indicate that single- culture (96uX well format). Specimen extract
exposure Ciguatect™ SPIA would produce a productsclassifiednon-toxicbythemousebioas-
falsenegativerateof76%andafalsepositiverate saywerefoundtopossesssodiumchanneleffects
of 54%. Triple-exposure Ciguatect™ SPIA at ID90dosing that ranged from 7 to40mg MTE.
would produce a false negative rate of45% and The latter finding, if considered in conjunction
a false positive rate of 43%, and the REM with mouse bioassay results from the ciguatera
Ciguatect™ SPIA would produce a false nega- casespecimenVI-108(i.e.,mousesurvivalat45g
tivTehreatCeiogfua3t9e%cta™ndSPaIfAalwseaspoesxitaimvienreadtefoorfi4n4te%r.- uMtTilEizaatniodneoxfpi9ra0tgioMnTatE90ign MthTeE)m,oussueggebsitosastshaayt
ference from potential fish decomposition may approximate a correlate forhuman toxicity.
IMMUNOBEADASSAYFOR BIOTOXINS INCARIBBEAN FISH 487
TABLE 5. Sodium channel activity ofspecimen solid-phase extraction products (ID90inhibitory dose which
reducescell viability by 90% at22hrexposure). Valuesexpressed in milligram muscle tissueequivalentsper
well;mousebioassaysurvivaltimes;Ciguatect SPIA scoresanddecomposition indicators. * indicates data
provided by Dr D.L. Park, University ofArizona, t indicates apparent inconsistency with regard to scoring
scales. #indicatesdoseof45g MTEadministeredtomice.
Specimen Sodium 90gMTE Ciguatect™score Decompositionproducts
IcDh9a0nn(emlg biomasosuasyeTD eSxipnogsluer-e eTxrpiopsluer-e REM* CadaUvge/rgine Hismtgam%ine
MTE/well) (hr) (SD) (SD)
VI-13 >40 >48 1.5(0.5) 0.2(0.4) 3,3 <0.5 0.1
>48
VI-59 32 >48 2.2(0.4) 2.7(1.1) 5,6f <0.5 0.1
>48
VI-63 22 >48 2.5(0.5) 2.7(0.7) 1.1 <0.5 0.1
>48
VI-32 20 >48 1.8(0.4) 3.8(1.2) 3,3 <0.5 0.1
>48
VI-81 9 >48 0.8(0.4) 1.0(0.0) 4,5 7.2 0.2
>48
VI-54 7 >48 1.8(0.4) 2.5(0.5) 3,3 <0.5 0.1
>48
VI-73 6 1.10 0.8(0.4) 0.0(0.0) 5,5 <0.5 0.1
4.66
VI-65 4 16.00 0.2(0.4) 0.3(0.5) 4,5 <0.5 0.1
>48
VI-53 2 2.50 1.8(0.4) 2.7(1.1) 3,3 8.4 0.2
6.40
VI-85 2 1.21 1.7(0.7) 2.0(0.6) 5,5 <0.5 0.1
1.61
VI-77 2 0.66 1.7(0.7) 1.2(0.4) 3,3 <0.5 0.1
0.63
VI-38 2 0.33# 0.5(0.5) 3.7(0.5) 5,5 <0.5 0.1
0.63#
VI-57 2 0.50# 1.0(0.0) 2.7(1.2) 5,5 3.7 0.1
0.45#
CONCLUSION Jester,MsDeliaClausen,DrSusanMcCarthy,Dr
Antony Guarino and Ms Kathy El Said forassis-
Ciguatect™ SPIA performance with cigua- tance in preparation of extracts, and reading of
toxic Caribbean finfish may be characterized by results. Forassistancein fishcollection wethank
low specificity rates and high false positive and Mrs Ann Seiler and staffofthe Division ofFish
falsenegativevalues. Extrapolatingtheseperfor- andWildlife,St.Thomas,U.S.VirginIslands,Mr
mance characteristics to a market situation im- Louis Greaux, President of Frenchtown-St.
plies that a proportion ofwholesome fish might Thomas Fishing Tournament, and Mr Mark
falselybe identified as ciguatoxic and an equally MarinandMrSidSmithoftheSt.ThomasSpear-
significant proportion of ciguatoxic fish might fishing Club. Appreciation is extended to Drs
reach the marketplace undetected. Conclusions Ron Manger, Jim Hungerford and Marleen
of the present study cannot be extended to Wekell,MsLindaLejaandMsSueLee, Seafood
Ciguatect™ SPIA performance with Pacific Products Research Center, FDA, Bothell, WA,
Ocean finfish (where the immunoassay for performing the bioassays for Na channel ac-
originated) without separate evaluation of the tivity.
method using fish from the Pacific region.
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