CA2080925A1 - Simplified solid-phase immunobead assay for detection of ciguatoxin and related polyethers - Google Patents

Abstract

The development of a highly-simplified solid-phase colored latex immunobead assay for the detection of ciguatoxin and related polyethers. This procedure was compared with a previously-reported stick enzyme immunoassay. Chi square analysis of two separate experiments 153 and 283 fish of various species gave X2 values of p < 0.001 and < 0.005, respectively. Agreement between the two procedures with 26 fish implicated in ciguatera poisoning was 100 %.
A preliminary assessment in the field showed encouraging results.
The procedure appears to be simple and applicable to field use.
Furthermore, this procedure should be applicable for other antibody-antigen detections, especially low dalton determinants.

Description

WO gl/16~31 PC'r/US~1/()2703 `: 2080~2~ i :
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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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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~

' . 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 .,,., , _ f~.
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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 ' ' ' ;'' . , " " ' "' ,," ,' ,', " ' ' ' "' ~O9l/16631 P~T/US9l/027~3 ~, ~ - -5- 208092~
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 .

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 ' ' ,, :' . ' 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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. ' .'. ' , ' ', ', ~ ' , , . ' . ' , ~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) . .; . -WO91/16631 P~/US91/~27~3 1~' -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) _ 15 ~ta~. 259 (89.0) (11) -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 --~---: 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 . . . f:~i 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 * 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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-13- 2080`92~ :
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.

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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Claims (21)

AMENDED CLAIMS
[received by the International Bureau on 28 August 1991 (28.08.91);
original claim 1 amended; new claims 2-21 added;
(3 pages)]

1. In a method for detecting ciguatoxin and structurally-related polyether marine toxins in fish tissue, including attaching the toxin to a support and fixing the toxin to the support, wherein the improvement comprises exposing the fixed toxin to immunobeads.

2. In a method as recited in claim 1, wherein the immunobeads comprise an antibody against the toxin attached to a colored, latex bead.

3. In a method as recited in claim 2, wherein the antibody is a monoclonal antibody.

4. In a method as recited in claim 2, wherein the colored latex bead is about 0.3 to about 0.4 µm in diameter.

5. A method for detecting the presence of ciguatioxin or related polyether marine toxins in fish, comprising the steps of:
attaching the toxin to a support;
fixing the toxin to the support with a fixer to form a toxin-support complex;
binding immunobeads to the toxin-support complex;
rinsing excess and unbound immunobeads from the support with a buffer; and quantitating the amount of immunobeads bound to the support.

6. The method as recited in claim 5, wherein the toxin is isolated by inserting the support into a contaminated fish.

7. The method as recited in claim 5, wherein the isolated toxin is fixed to the support by immersing the support into absolute methanol.

8. The method as recited in claim 5, wherein the support comprises a solid member coated with a toxin adsorbing material.

9. The method as recited in claim 8, wherein the solid member comprises a bamboo stick.

10. The method as recited in claim 8, wherein the toxin-adsorbing material comprises LIQUID PAPER.

11. The method as recited in claim 5, wherein the immunobeads comprise an antibody against the toxin attached to a colored latex bead.

12. The method as recited in claim 12, wherein the antibody is a monoclonal antibody.

13. The method as recited in claim 12, wherein the colored latex bead is about 0.3 to about 0.4 µm in diameter.

14. A kit for detecting the presence of ciguatoxin or related polyether marine toxins in fish, comprising:
a support for binding toxin;
a fixer for fixing toxin to the support;
an immunobead suspension for assaying toxin bound to the fixed support: and a buffer solution for washing the fixed toxin-bound supports after they have been contacted with the immunobeads.

15. The kit as recited in claim 15, wherein the support comprises a solid member coated with a toxin-adsorbing material.

16. The kit as recited in claim 16, wherein the solid member comprises a bamboo stick.

17. The kit as recited in claim 16, wherein the toxin-adsorbing material comprises LIQUID PAPER.

18. The kit as recited in claim 15, wherein the fixer comprises methanol.

19. The kit as recited in claim 15, wherein the immunobead comprises an antibody against the toxin bound to a colored latex bead.

20. The kit as recited in claim 21, wherein the antibody is a monoclonal antibody.

21. The kit as recited in claim 21, wherein the colored latex bead is about 0.3 to about 0.4 µm in diameter.