Note: Descriptions are shown in the official language in which they were submitted.
WO gl/16~31 PC'r/US~1/()2703
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10SIMPLIFIED SOLID-PHASE XMMUNOBEAD ASSAY FOR
DETECTION OF CIGUATOXIN AND REL~TED POLYETHERS
- 'ition
: The invention relates to the use of antibodies
for the detection of ciguatoxin in ~ish.
.,
Background of the Invention
Immunological analysis o~ ciguatoxin (CTS) and
related polyethers by radioimmunoassay (RIA) using
I~25-labelled sheep anti-CTX prepared by immunization
of sheep with puri~ied CTX coupled to human serum
albumin (HSA) was initiated in 1977. (See Hokama et
al., "A Radioimmunoassay for Detection of Cigua-
' toxin,i~ oxlcon, 15, 317-215 (1977), incorporated
i~ ~ 25 herein by this reference~. This RIA was used to
', :screen several thousand Seriola dumerili (kahala,
amberjack) in a two-year study with the United
Fishing Agency in the State of Hawaii. No false
; nsgatives were reported, with 15% of the total fishes
examined rejected as potentially toxic. Subse-
: quently, an enz~me immunoassay procedure was
d~veloped using the same sheep-anti-CTX labelled with
horseradish pe:roxidase for testing fish tissues.
(See Hokama et al., "A Rapid Enzyme Immunoassay (EIA)
for the Detection of Ciguatoxin in Contaminated Fish
Tissues," Toxicon~21, 817-824 (1983) and "An Enz~me
I~munoassay :for the Detection of Ciguatoxin and
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WO91/16631 PCT/US91/02703
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2~809 ~ Competitive Inhibition of Related Natural Polyether
Toxins," Seafood Toxins, ACS Symposium Series 262,
Washington, Dc 307-320 (1984); Kimura et al.,
"Comparison of Three Different Assays for the Assess-
- 5 ment of Ciguatoxin in Fish Tissue: Radioimmunoassay,
Mouse Bioassay and ln vitro Guinea Pig Atrium Assay,"
Toxicon 20, 907-912 (1982), and U.S. Patent No.
4,816,392 to Hokama, all incorporated herein by this
reference. ? This procedure per~itted the analysis of
cross-reactivity of puri~ied ciguatoxin with purified
okadiac acid (OA), brevetoxin (PdTx), maitotoxin
(MTX), and monensin. A radioimmunoassay procedure
for PdTx also demonstrated the cross-reactivity of
CTX and PdTx. (See!Baden et al., "Cross-reactivity
~- 15 in Immunoassays Against Toxins Isolated from
Ptychodiscus brecis," Toxic Dinoflaqellates, Elsevier
Applied Science Publishers, NY, 363-368 (1985),
incorporated herein by this reference.~
Development of a simplified stick enzyme immuno-
assay (S-EIA) was initially reported by Y. Hokama
(see "A Rapid Simplified Enzyme Immunoassay Stick
Test for the Detection of Ciguatoxin and Related
Polyethers from Fish Tissues," Tox icon,_23, 939-946
(1985), incorporated herein by this reference), using
the sheep-anti-CTX. An extensive study reported
recently using the S-EIA procedure gave no false
negatives in the test system using monoclonal anti-
body to TX, MAb-CTX. This procedure proved useful
inn the laboratory, but was found to be impractical
in the field and onboard ships.
I have now developed a highly simplified method
using colored latex beads to which the MAb-CTX is
bound. Fish-tissue toxin bound to liquid-coated
stick colors when immersed into suspensions o~ latex~
MPb~CTX reagent~
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. WO91~16631 PCT/US91/02703
. ~3~ 2080925
1 The present invention presents the initial
development of the procedure and the examination of
various toxic and non-toxic fishes in comparison with
the sti.ck enz~me immunoassay.
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WO 91/16631 PCr/USgl/02703
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20809~
l petailed Descri~tion
Source of fish: Fish samples were obtained from
various sources. The fishes implicated in ciguatera
poisoning were obtained from outbreaks in the State
o~ Hawaii, Philippines, CAlifornia, and Texas The
clinical symptoms o~ the patients invoked were
characteristic of what is categorized as ciguatera
poisoning. (See Bagnis et al., "Clinical Features on
12,890 Cases o~ Ciguatera Fish Poisoning in French
Polynesia," Proqress in Venom and Toxln Research, P.
Gopalakrishnakone, C.K. Tan, eds., Kent Ridge,
Singapore, 272-384 (1987); Hokama et al., "Ciguatera
Poisoning: Clinical and Immunological Aspects," J.
Toxicol:~ Toxin Review, 5, 25-53 (1986); and Kodama
et al., "Ciguatera Poisoning: Variation in Symptom-
ology," Toxicon~ 27, 593-595 (1989), all incorporated
herein by this reference~) Identification of the
fishes were determined according to Tinker, S.P., "A
Handbook of the Marine Fishes of Hawaii and the
Central Pacific Ocean, Honolulu" Fishe _ of Haw ii,
Hawaii Service, Inc., 1978, incorporated herein by
- this reference.
Monoclonal_ antibodies (MAbs~ : The method of
Schrier et al., Hyb~r_doma Techniaues~_~old Harbor
Laboratory, Cold Spring, NY, 1980, incorporated
herein by this reference, based on the original
report of Kohler et al., "Continuous Cultures of
Fused Cells Secreting Antibody of Predefined Specifi-
city, 1I N ure, 156, 494-497 (1975), incorporated
herein by this reference, was used. Hybridoma
preparation for Mab-CTX was carried out using puri-
fied CTX according to methods of Hokama et al.,
"Assessment of a Rapid Enzyme Immunoassay Stick Test
for the Detection of Ciguatoxin and Related Polyether
Toxins in Fish Tissues," Biol. Bull~_ 172, 144-153
(1987; "Monoclonal Antibody (MAh) in Detection of
Ciguatoxin (CTX) and Related Polyethers by the Stick
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~O9l/16631 P~T/US9l/027~3
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1 Enzyme Immunoassay (S-EIA) in Fish Tissues Associated
with Ciguatera Poi60ning," Mycotoxins and Phycotoxins
'88, The Netherlands, Elsevier Applied Publishers BV
Amsterdam, 303-310 (1989); and l'Monoclonal Antibodies
in the Detection of Ciguatoxin and Other Toxic Poly-
ethers in Fish Tissues by Rapid Poke Stick Test,"
Proc. _5th Int. Coral Reef congress, 4, 449-474
(1985), all incorporated herein by this reference.
Stick Enzym _ Immunoassay (S-EIA): The method
for assessment of the fish samples run in conjunction
with the new procedure was the S-EIA previously
reported.
Solid-~ase Immunobeadj~ y: The solid phase
consisted of a paddle made of a bamboo stick coated
with organic base solvent correction fluid supplied
by Pentel of America, Ltd., Torrance CA 905034.
Immunobead consisted of blue colored latex, 0.314 ~
in diameter, supplied by Seradyn, Inc., Particle
Technology Division Ind., IN.
Optimization of_SPI As.~y: Various parameters
were examined to eliminate non-specific binding of
colored latex to a coated bamboo paddle. Various
concentrations of MAb-CTX with a constant suspension
of colored latex were attempted to give the best
specificity and sensitivity. Experiments were per-
formed with colored latex alone in suspension (1%
wt/wt in PBS buf~er). Various MAb-CTX concentra-
tions ranging ~rom 0.05, 0.075, 0.10, 0.13, 0.20,
0.45, 0.50, to 1.0 mg/ml were added to the 1% colored
latex. The liquid paper-coated sticks were examined
as-is or were coated with a 1% wt/wt solution of
Human Serum Albumin (HSA), then examined as non-Me-
QH-fixed or NeOH-fixed with the MAb-CTX-colored late.
MAb-CTX was coated onto liquid-paper sticks and then
examined with MAb-CTX-color lat~x.
The optlmum condition obtained from these
experiments was used for examining toxic fish rrom
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WO91/16631 Pcr/ US91/02703
-6- t
~ 8 0 9 ~ 5 the Depar~ment of Health (State of Hawaii) and toxic-
fish samples from elsewhere which had been implicated
in ciguatera poisoning. Raef fishes from various
sourcPs were also examined. For the development of
the optimum conditions, known t;oxic fishes implicated
in ciguatera poisoning were employed (for the posi-
tives). Negative controls were protein-coated sticks
fixed with MeOH or unfixed blank sticks. The method
according to Singer et al., "The Latex Fixation Test:
One Application to the Serological Diagnosis of the
Rheumatoid Arthrltis," Amer. J ~Med., 888-892, Dec.
(1956), and incorporated herein by this referenoe,was
used for the"~preparation of the immunobeads. The
optimum concentration of MAb-CTX protein was 0.45
mg/ml of the 1% bead suspension. This is designated
as the immunobead. The immunobead was mixed thor-
oughly before use.
Solid-phase Immun_assay l SPIAL: An inch-deep
incision is made into the filet portion of the fish
near the head region, as shown below The coated
paddle end is inserted into the incision and pushed
up and down to touch the fish tissue. The paddle is
removed, air-dried, and fixed quickly (1-3 s~conds)
with absolute methanol. After air drying, the coated
methanol-fixed end of the paddle is immersed into 0.5
ml of the immunobead color suspension. After 2, 5,
and l0 minutes, the stick is examined and washed in
saline. Any fish giving the stick a distinct colora-
tion after 5 minutes is considered positive. If
negative, immersion is continued up to lO minutes.
After l0 minutes, no color, very diffuse color, or no
distinct coloration oP the paddle is read as nega-
tiveO In this case, the same procedure is repeated
with another stick from another area of kha same
~ish. If the stick is negative after lO minutes, the
fish is considered safe to eat. A borderline raading
t+, +) in two sticks after their lO-minute readings
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WO~/16631 PCT/US91/02703
2~8~92~ 1
1 should not be eaten. Similarly, any single stick
reacting in 5 minutes or less, read as + -t, should
not be eaten. Examples are given in the results.
Control blank sticks (untreated coated stick, nega-
tive) should be run in paralle:L with the fish samplesticks. Similarly, a known positive (implicated in
toxicity) should be run with the unknowns. The
concept and methodology are il:Lustrated below.
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S PIA CO NC E PT
>'
2 0Slich Insorl inlo lish lissuo
l l l l > Tolin moloculo
_ ~ ~ 1 _ . _¦ )k Uonoclonal anllbody
I . I l~bollod wilh Ir~lex
2 5 > ~ l 3~1 Color oi lalex all~chcd lo ~l~o
. > J _ ~ anllbody indic~los pO5~1 ro hsn
ril ~n mo~hyl olcoholIncubalo in anllbody
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20 W 91/l6631 -8- ~Cf/U~')l/02703
1 Comparison~ h SPIA
a. Department o~` Health-implicated fish in
clquatera poisonin~:
Table l shows the species, source and the test
values given by the S-EIA and SPIA procedures for
fish implicated in ciguatera poisoning. All samples
were obtained from the Department of Health, State of
Hawaii, except for two specimens (Antigua and Cali-
fornia). The S-EIA values were all in the rejection
category and in complete agreement with the SPIA
results. The majority of the toxic fishes were from
the Big Island of `Hawaii and associated with the
Caranx sp. ~
Table l. Fish I~plicated in Ciguatera Po ~ ning from the
D~xrbY~ of Health, State of~waii and
El~ere~ ~rison of S-~IA and SPIA
~ . . . _ . . _ _ _ _
Species of Fish ~o~e Test Values
S-EIA SP~
20 C~x sp. Big Island +
C~x sp. Big Island +
Caranx sp. Big Island +
C~x~x sp. Big Island +
C~x sp. Big Island + +
Caranx sp. Big Island + +
C~x sp. Big Island + +
25 ~x sp. Big Island + +
C~R~X sp. Big Island + +
C~x~x sp. Rig Island + +
C~x sp. (~lu) Big Island + +
Ca~Lx sp. Big Island + +
C~ sp. Big Isl~nd + +
~x sp. (Papio) Big Islc~nd ~ +
C~nx sp. Big Island + +
30 C~x sp. Big Islan~ + +
~ sp. Big Island -1- *
Seriola sp. Oahu + +
Seriola sp. Oahu ~ +
Seriola sp. Oahu -~ +
Cq~lopholisc~s Big Island -~ +
C~lopholisc~~ Oahu + ~
35 Mhlloidich~h~s al~i~ Molokai + +
Lutianus sp. A~tigua + +
~ oahu *
Sphyr_ena California + +
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WO91/16631 PCT/US91/02703
~9~ 208~925
1 TJ~l of 26 implica~ed fish all ~ rejection (~ or ~) in
both S-~ an~ SPIA.
b. Results of Routine Ru~ns Examined ln Compari-
son with the ~Stick-EnzYme ~mmunoassay (S-EIA):
An initial study of 153 fish, mostly ~acks
(ulua) and amberjacks (kahala) was compared by the S-
EIA and the newly-established SPIA. The chi square
(X2) for the 153 fish samples was p < O.OOl, suggest-
` lO ing a good association between the two tests. Eighty
percent of the 153 samples were in agreement between
the two tests, and 20% were in disagreement. The
SPIA appeared to be more sensitive than the S-EIA.
That is, most of the samples (12~ were negative with
15 the S-EIA and positive with the SPIA, while 8%
represented the inverse (S-EIA+/SPIA-).
A second ~omparison of the S EIA and SPIA on 283
; ~ishes gave essentially the same X2, which was equiva-
lent to p < 0.005 for all fish. Eiyhty-three percent
20 of the 283 samples were in agreement between the two
tests, while 17% were in disagreement. Again, the
SPIA appears to have a yreater sensikivity than S-
EI~, with many of the samples (15%) negative in the
, S-EIA, and positive with the SPIA, while 2% repre-
e 25 sented the inverse ~S-EIA+/SPIA-). The x2 value of p
< 0.005 suggests a significant association between
the two tests.
- Analysis of some individual species from Study
2 is shown in Table 2 in the comparison o~ the S-EIA
30 and SPIA tests. Fish giving good agreements between
tests are general~y carnivorous (ulua, kahala,
Luttanus sp., and wahanui). The greatest disagree-
ment is seen with the halalu (mackerel). In most of
the disagreements, the SPIA appears top be the more
sensitive (-S-EIA/+SPIA).
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WO 91/16631 PCr/lJS91/02703
2~0925 -lO-
- 1 ~ble 2. Campar~son of ~he Stic~-Enzyme ~=n~say (S-ELA)
and Solid Ph~se ~ad (SPL~) with Various
Species o~ Fish
Species ~ al Assay Data
~ S-E~ySPIA
Agree Disagrae
+/+ and -/- (%) -/+ or +/ (%)
_ _
. 1. ~ sp. 1~:7 110 (94) 5 2 (6)
(Ulua, Papio)
25 1i 9 9 (100) 0 0 (O)
(K~hala, ~ack)
3. F~mily r~gil dae 21 20 (95) 1 0 ~5)
(k~let)
4. I~a~ 10 ~ (40) 6 0 (60)
1 ~ c~ ,men~hthalcmws
(Halalu, Big Eye Scad Fish)
5. ~hlia sar~vi~s 12 8 (67) 4 0 (33)
(Flagtail Fis21, Aholehole)
6. ~etus ~g 39 (80) 10 0 (20)
2 0 striaosus
~Kole, Sur~gonfish)
7. ~carlthun~s sp. 23 20 (87) 2 1 (13)
(~, ~nini, M~iko)
. IIItianu~; ka~nira 5 5 (100) o o (o)
(Taa~e, Sna ~ )
9. Mvris~ristis ~p. 5 5 (lOO) o o (0)
(Mer~achi, Squ:i~ Fi~)
lO. Bodiarms sp. 4 2 (50) 2 0 (50)
(Aawa, W~ass~)
11. ~hareus fur~atus 4 4 (100) 0 ()
(Wahanui, Bla~ Forktail Snapper)
12. SFihyraera sp. 4 3 (75) 0 1 ~25)
, Barra~l)
13. P~aF~eu~orphyreus 3 2 (67) 1 0 (33)
(~, Goat~i
14. I~tiarms flayl~s 3 3 (100) 0 ()
(i~ au, Sna~pex)
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WO91/16631 P~/US91/~27~3
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-11 , 208092
. 1 Table 2, continued . . .
i
Species T~ Assay Data t
N~ S-E~ySPIA
~x~ Dlsa~
~/~ and ~ or +/- (%)
15. Iutianus sp. 3 2 (67) 1 0 (33) '
(P~i3d ~apper)
10 16. Mhlloidicht~ys 2 1 (~0) 1 0 (50)
auriflan~na
(Ike, Goatfish)
17. Misoellan~ous 6 4 (67) 1 1 (33)
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15 ~ta~. 259 (89.0) (11)
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Table 3 summarizes the cQmparison of S~EIA and
SPIA of the two studies, totalling 436 fishPs.
There is an overall 80% agreement, with 20% dis-
agreement (12% = -S-~IA/+SPIA, and 8%
~S-EIA/-SSPIA). The 20~ samples of fish in categ-
ories -/+ and +/- will be further examined follow-
ing extraction and then chromatographed by silica
gel, with final assessment of the fractions by
guinea pig and mouse bioassay.
Table 3. Summary of Comparison of S-EIA and SPIA
~ Data on Same Fi~h Samples
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: Total Fish Results of Tests
S-EIA/SPIA
Agre~ Disagree
~/~ and -/- ( % ) -/+ or +/~
436 349 (80) 51 36 (20)
WO91/16631 I'CI/U591/027~3
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208092~ - -12-
1 * Twelve percent of the disagreement is in the
S-EIA negative and SPIA p~siti~e group, while
the remainder is in the +/- group.
Table 4 shows the preliminary data obtained by
a voluntary sportsfisherman assessing the solid-
phase immunoassay presented earlier. A study byMr. Dale Takata of the Honolulu City Water and
Waste management is summarized Mr. Takata volun-
teered to use the new SPIA test. 0~ the 55 fishes
of various species caught, 37 negative and border-
line ~ishes were eaten, and 18 were rejected as
- being positive,~ Consumption of two fishes tpapio
and kole) from the positive group caused ciguatera
poisoning in two individuals who were warned of the
fish's toxicity based on the SPIA test.
Table 4. Biy Island (Waikoloa) Study with the
Solid-phase Immunobead Assay
Total Number of Fish Number %
- - _
Positive
1~ 33
Negative or Borderline
37 67
* 2 of the 18 positive category fish were con-
sumed, and both individuals showed ciguatexa
poisoniny symptoms in less than 2 hours after
eating. The fishes involved were C. striqosu~s
and Caranx sp. (Papio).
An encouraging preliminary study strongly
suggests the applicability o~ the SPIA for field
use. This wsuld be a significant advancement ~or
reef sportsfishermen, long-distance yachters, and
perhaps in-shore commercial fishermen.
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WO91/16631 ~CT/US91/02703
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l Comparative assessment of the S-EIA and SPIA
shows very good association, with the Department o~
Health-implicated fish showing a 100% agreement.
However, comparison of a variety of fish in two
separate studies demonstrated an agreement of 80%
(with moderate) association between the two tests.
This is in part due to ma~or difference in some
species. For example, a great discrepancy is shown
for the mackerel (halalu). Fo:rtunately, this
species has not caused ciguatera poisoning in
Hawaii, and therefore, the dif~erences may be due
to non-specific binding on the part of non-specific
binding on the part o~ the SPIA test. (In general,
this test appears to be more sensitive than the S-
EI~.) Nevertheless, in the two major speciesimplicated in ciguatera poisoning, Caranx sp. and
Caranx sp. (Papio). Seriola sp., the association
appears to be very good between the two tests.
(See Table 2.) Similarly, the association of the
two tests with other carnivorous species appears to
be good. The S-EIA procedure has been thoroughly
tested the past three years.
Further examination following extraction of
the fish samples showing discrepancies such as S-
EIA-/SPIA+ or S-EIA~/SPIA- will be attemp~ed by the
guinea pig atria (see ~iyahara et al., "Pharmaco-
logical Characterization of the Toxins in Cugateric
Fishes," MYCOtOXinS and Phycotoxins '88, Elsevier
Science Publishers B V Amsterda~, The Netherlands,
399-406 (19B9), incorporated herein by this re*er~
ence) and mouse assay.
WO91/16631 PCT/US91/02703
208092~
l The single volunteer study gave promising
results, and several more individuals and fishing
clubs are being mobilized for an extensive evalua
tion. Refinement of the SPIA is still in progress.
It is hoped to achieve a goal for wide use of the
SPIA where ciguatera poisoning is endemic.
Furthermore, the SPIA method described should be
applicable to other antigen-antibody systems, espe-
cially when the antigen is an epitope or hapten of
low molecular wèight.
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