I AN IMMUNOLOGICAL APPROACH FOR CTX

by Y. Hokama', L.H. Kimura', K. Shiraki', of study such as tissue distribution, synthesis and R. Shomura2, R. Uchida2, B. lto2, in some cases the structure and function of the B. Takenaka3, and J. Miyahara4 haptenic molecule. The application of the sensitive immuno- I NT RO D UCTl0 N logical methods for the detection of marine The application of immunological techniques toxins, such as saxitoxin, ciguatoxin, and tetrado- for the detection of low molecular weight, non- toxin, merits strong consideration in light of the immunogenic compounds has increased markedly minute amounts of these toxins in the natural in the past decade. The ability to conjugate these environment. The minute amount in tissues has haptenic small molecules covalently to appro- constituted serious hazards to the consumer and priate immunogenic carriers via their functional has created anxiety within the fishing and shell- groups has led to the production of specific anti- industries. bodies to the haptenic molecules following This study discusses the immunological administration into appropriate . Primary approaches for the development of a radio- functional groups or moieties amenable to conju- immunoassay procedure for the detection of gation, provided they are accessible to chemical ciguatoxin (Hokama et al., 1977). The method- coupling, include amino, hydroxyl, and carboxyl ology presented is applicable for the development groups. Some common methods for coupling of immunological procedures for the other low include carbodiimide, nucleophilic substitution, molecular weight marine toxins such as saxitoxin, diazo and azide coupling. In some cases, for tetradotoxin, and Gymnodinium breve toxins. In example with lipid antigens, noncovalent com- addition, for correlative analysis, the guinea pig plexes have been utilized using methylated bovine atrium procedure (Miyahara et al., 1979) for serum albumin as the carrier (Butler et al., 1973; quantitation of ciguatoxin and maitotoxin from Becher, 1976). crude extracts of is also included in this The production of a specific antibody to the study. Comparison of the RIA procedure with the low molecular weight compounds can then be mouse M.U. test is also presented in this study. utilized for the development of sensitive and specific immunological procedures such as radio- METHODS FOR CIGUATOXIN immunoassay (RIA) and enzyme-linked immune absorbent assay (ELISA) for detection of minute AN ALY S IS amounts of antigens or antibodies. In addition, The development of the RIA procedure for the antibodies are useful in examining other areas the direct detection of ciguatoxin (CTX) from

lDepartment of Pathology. John A. Bums School of Medicine, University of Hawaii at Manoa; 2Honolulu Laboratory, National Marine Service, NOAA, US Department of Commerce; 3United Fishing Apncy, Ltd.; 4Dewtment of Pharmacology, John A. Burns School of Medicine, Uniwnity of Hawaii at Manoa.

I_ 2 fish tissues has been reported previously (Hokama some instances. Toxic and nontoxic fish tissue et at., 1977). samples are examined with each set of unknown Purified ciguatoxin (Scheuer et al., 1967) determinations. isolated from livers of toxic eels was conjugated The present study includes examination by by the carbodiimide procedure (Oliver et al., the RIA procedure of fishes recovered from clin- 1968) to human serum albumin (HSA). The latter ically diagnosed ciguatera poisoning cases and is the carrier for the lipid hapten-ciguatoxin. nontoxic fishes from commercial sources. The Precipitation of the ciguatoxin-HSA (CTX-HSA) toxic fishes were obtained through the courtesy with cold acetone indicated that CTX was in the of the Hawaii State Department of Health. The precipitate (CTX-HSA) and not in the acetone nontoxic fish samples were obtained from various phase. This suggested that most if not all CTX commercial and noncommercial sources. Reef was bound to HSA. fishes from Tahiti were obtained through the cour- The antibody to CTX-HSA was raised in tesy of Dr. Bagnis of the lnstitut de Recherches sheep given three weekly subcutaneous injec- Medicales Louis Malarde. tions of a CTX-HSA-Freunds complete adjuvant A correlative study of the RIA procedure and mixture (1:l). A total of 500 pg of CTX and the in vitro guinea pig atrium assay was carried 50 mg of HSA (CTX-HSA) was administered to out for several samples of toxic and nontoxic the sheep in a period of 7 weeks. The sheep was fish tissues. The details of the guinea pig atrium bled 10 days after the last injection and the serum analysis were reported recently by Miyahara removed and stored in 30 ml aliquots at -20°C. et al. (1979). The crude extracts of the fish tis- The details of the determination for the pres- sues were prepared according to a previously ence of anti-CTX-HSA by immunoelectrophoresis described procedure (Yasumoto et al., 1977). A and mouse studies and for the development of the 20-9 tissue is homogenized and extracted with RIA procedure have been described (Hokama et 200 ml hot methanol. The residue is removed by al., 1977). In brief, the RIA method consists of filtration and the methanol phase concentrated to the following steps: (1) sample tissues from the 10 percent of the original volume. The concen- dorsal and ventral muscles, tails, or gonads of the trated solution is diluted with distilled water fish; (2) dehydrate samples in oven at 70°C for (5 volumes) and the lipid solutes extracted with 1 hour; (3) after cooling, weigh 15-mg samples; diethyl ether. The ether is evaporated and the (4) rehydrate with buffered saline; and then crude lipid residue dissolved in minimal methanol remove buffer; (5) add 0.2 ml of 1z51-labelled and stored at -20°C until ready for study. Assay sheep anti-CTX-HSA (sp. act., 1.03 mCi/mg for the mouse toxicity procedure has been protein) containing 50,000 cpm/O.l ml; (6) shake described previously (Yasumoto et al., 19711. for 3 hours at room temperature; (7) add 3.0 ml Based on the examination of fishes from buffered saline; (8)aspirate buffer thoroughly; human ciguatera poisoning and previous results and (9) count the sample in gammacounter. obtained by RIA (Hokama et al., 1977) the fol- After background correction, the cpm/tube is lowing arbitrary criterion has been established divided by 15 mg to give cpm/g tissue. Samples relative to the cpm/gm tissue values. are determined in triplicates, or duplicates in Cpm/gm Tissue Toxicity Index Volume 2, Number 1 ISSN 0199-1 37~ < 350,000 Negative SEA GRANT QUARTERLY 350,001 to 399,999 Borderline published quarterly by > 400,000 Toxic UNIVERSITY OF HAWAII SEA GRANT COLLEGE PROGRAM The diagnosis of ciguatera poisoning of 2540 Maile Way, Spalding Hall 255 patients following consumption of suspected toxic fishes is based on the clinical symptoms "."CIII,**W." Honolulu, Hawaii 96822 described previously (Okihiro et al., 1965; Bagnis, Second-Class postage paid at Honolulu, Hawaii 1968). POSTMASTER: Send address changes to Sea Grant Quarterly, 2540 Maile Way, Spalding Hall 255, RESULTS Honolulu, Hawaii 96822 RIA Examination of Fish Samples. The results Rose Pfund, Editor Jack R. Davidson, Director of the examination of individual cases of fish sam- ples recovered from clinically diagnosed ciguatera 3

Table 1, RIA Examination of Fish Samples From Clinically Documented Ciguatera Poisoning

No. of RIA Results Fish Common/Hawaiian Name Remarks Samples CPM/GM Tissue 3 Scarus sp. Parrotfishhhu 463,039 Toxic Scarus sp. Parrotfishh hu 524,106 Toxic Scarus sp. Parrotfishhhu 421,064 Toxic 1 Elagatis bipinnulatus Rainbow runner/kamanu 463,636 Toxic 6 dumerilii Amberjackkahala 41 5,870 Toxic Seriola dumerilii /kahala 549,534 Toxic Seriola dumerilii Arnberjacklka hala 41 2,600 Toxic Seriola dumerilii Amberjackkahala 447,045 Toxic Seriola dumerilii Amberjacklkahala 436.778 Toxic Seriola durnerilii Amberjackkahala 296,667 Toxicity not detected by RIA 13 or Carangoides sp. Jackhlua 41 4,489 Toxic Caranx or Carangoides sp. Jackhlua 420,667 Toxic Caranx or Carangoides sp. Jackhlua 492,634 Toxic Caranx or Carangoides sp. Jack/ulua 505,779 Toxic Caranx or Carangoides sp. Jack/ulua 497,406 Toxic Caranx or Carangoides sp. Jackhlua 400,867 Toxic Caranx or Carangoides sp. Jack/ulua 711,346 Toxic Caranx or Carangoides sp. Jackhlua 448,936 Toxic Caranx or Carangoides sp. Jackhlua 351.400 Borderline Caranx or Carangoides sp. Jackhlua 387,553 Borderline Caranx or Carangoides sp. Jack/ulua 362,920 Border Ii ne Caranx or Carangoides sp. Jackhlua 370,597 Borderline Caranx or Carangoides sp. Jackhlua 300,300 Toxicity not detected by RIA 2 Mugil cephalus Mullet/ama'ama 449,967 Toxic (viscera sample) Mugil cephalus Mullet/ama'ama 462,753 Toxic 1 Aprion virescens Grey-snapperluku 482.1 33 Toxic 1 Monotoxis grandoculis Porgy/mu 660,556 Toxic 1 Priacanthus cruentatus Red big eye/aweoweo 382,584 Borderline 1 Lutjanus gibbus Paddle-tailsnapper 442,000 Toxic 3 Epinephelusmario Sea bass 479,339 Toxic Epinephelus rnario Sea bass 490,392 Toxic Epinephelus mario Sea bass 375.1 89 Borderline poisoning are indicated in Table 1. A variety of Table 2. Summation of RIA Values for Fishes From species has been implicated, including the fol- Clinically Defined "Ciguatera Poisoning" Cases lowing species: Scarus sp., parrotfishluhu; Elagatis and Nontoxic Fishes bipinnulatus, rainbow runner; Seriola dumerilii, amberjacklkahala; Caranx and Carangoides, sp., jac kluha ; Mugil cephalus, mu I letlama'a ma ; CPMlGM Tissue Aprion virescens, grey snapperlu ku; Monotoxis Fish (Mean * 1 S.D.) grandorulis, mu; Priacanthus, sp., red big eye/ Nontoxic Toxic P* aweoweo; Lutjanus gibbus, paddle-tail snapper; and Epinephelus rnario, sea bass. Two samples, a Several Speciest 264,202 434,654 C 0.001 Seriola durnerilii and a Caranx sp., gave RIA * 44,291 f 110,184 values in the nontoxic range. The mean 2 1 S.D. (n = 91) (n = 32) (434,654 f 110,184) value for the 32 toxic ~ samples is summarized in Table 2. This value is *p value based on two-tailed analysis. tSee Table 1 for the individual species listed for the toxic group; significantly (p < 0.001) different from the mean the nontoxic fishes in this category were sampled from the SBmB f 1 S.D. (264,202 k 44,291) value of the 91 species implicated in the "ciguatera poisoning" or the toxic goup. 4 non-toxic samples of essentially the same species and inotropic effect of ciguatoxin on guinea pig shown in Table 1. atrium is presented in Figure 2 with the r value Comparison of the RIA with the Mouse Tox- shown in Table 3 (r = 0.88). Although the sample icity, M.U. Figure 1 summarizes a comparative number is small, a significantly good correlation is shown between the RIA and guinea pig atrium assay. The latter assay appears to be a valuable method for ciguatoxin studies in correlation with the RIA and other biological procedures.

. :.. '

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0 05 IO I5 20 25 30 33 INOTnOCIC VILLI€

Figure 2. Comparison of the RIA procedure for cigua- toxin detection and the inotropic effect on 1 I I 0 10 20 30 guinea pig atrium. The inotropic values indica- MOUSE UNIT IMU) ted are obtained by dividing the increase in amplitude (inotropic effect) by the base line Figure 1. Cornparison of RIA procedure for ciguatoxin contraction. detection and the mouse toxicity based on M.U.; each circle represents a sample. (RIA DISCUSS ION values are average of triplicate samples.) A general approach for the use of immuno- study of the RIA with the mouse toxicity of reef logical procedures for application to small mole- fish tissues obtained from Tahiti. The incidence cules is presented elsewhere (Hokama et al., of ciguatera poisoning is high in French Polynesia 1980). The significant factors include: (1) the in contrast to other areas of the Pacific. The coef- importance of coupling of the low molecular ficient of correlation, r, is moderate between weight compounds to appropriate antigenic car- R1A:M.U. The r values are summarized in Table 3. riers, that is, presentation in positions of accessi- The R1A:Mongoose r value from a previous report bility, without gross changes in the haptenic (Hokama et al., 1977) is also shown in Table 3. molecule; and (2) selection or choice of the Comparison of RIA with Guinea Pig Atrium antigenic carrier. Of equal importance is the Assay. This correlative study between the RIA choice of for the production of antibody and the mode of administration of the hapten- Table3. Summation of the Comparison of the RIA carrier conjugate. The analysis and development Procedure with the Other Biological Methods of the immunological methods for the antibody for the Evaluation of Ciguatoxin raised will depend on the levels of the antibody and the haptenic molecule to be examined ~ NO.of System Coefficient of (Landsteiner, 1945; Rose and Friedman, 1976). Samples Correlation (r) Reference Essentially following the general procedures 66 R1A:M.U. 0.56 Hokama et al.. 1980 presented elsewhere (Hokama et al., 19801, devel- (mouse) opment of an immunological procedure for the 66 RIA:Mongoose 0.56 Hokama et at., 1977 detection of ciguatoxin (a lipid hapten) has been 13 RIA:Guinea pig 0.88 Hokama et al., 1980 initiated earlier (Hokama et al., 1977). This atrium study presents further evaluations of the RIA procedure by examining various species of fishes M.U. = Total weight in gm of mice killed within 48 hours by a 1 ml extract from 20 gm of fish tissue (Yasumoto et al., including those specimens implicated in ciguatera 1971). poisoning. 5 The comparative examination of fish samples evident in a recent report (Morris, 1980) from from Tahiti with the RIA and mouse (M.U.) pro- the Center for Disease Control, Atlanta, Georgia. cedure gave an r value which appeared to be Ciguatera poisoning has been reported as the similar (Table 3) to that demonstrated earlier for leading cause of poisoning from marine foods in RIA and mongoose. The lack of a better correla- the United States, surprisingly even more so than tion may be due to the highly sensitive nature of paralytic shellfish poisoning (Morris, 1980). the RIA test as discussed previously (Hokama et al., 1977). The capability and limitations of the biological procedures have been discussed earlier by other investigators (Banner et al., 1960; Banner et a1.,1961; Kosaki et al., 1968). The present state of the RIA procedure is significantly sufficient to distinguish toxic from nontoxic fishes. Furthermore, the test appears Figure 3. Gambierdiscus to be helpful for the examination of various species of fishes from different geographical areas for the evaluation of the presence or absence of toxic fishes. For example, fishes from Tahiti tended to have a higher incidence of toxic levels and cpm/gm tissue than similar species SUM MARY caught in Hawaii. (Compare data on fishes from The steps involved in the utilization of immu- Tahiti in Figure 1 with data on fishes in Hawaii nological procedures to study small molecules in Table 2.) Thus, in its present state the RIA pro- including marine toxins are as follows: (1) cedure can be utilized for the exploration of the conjugation of the small molecule to appropriate levels of toxicity and distribution among various antigenic carriers; (2) immunization of animals fish species in. different geographic areas, espe- for the production of antibodies; and (3) devel- cially where ciguatera poisoning is endemic. This opment of an immunological method for analysis would aid in the development of the fishing of the small molecule. The development of the industry. radioimmunoassay for ciguatoxin was carried out The limitations of the RIA procedure for accordingly. The RIA procedure for detection of wide-scale use at present include: (1) restrictions ciguatoxin showed moderate correlation with the on the number of sampling per day by a technol- mouse M.U. (r = 0.56) test. These results were ogist; and (2) a minimum of approximately 6 similar to that reported for the RIA and mon- hours required for the completion of the test. goose (r = 0.56) test. A good correlation of the Other difficulties include: (1) sampling error, RIA with the in vitro guinea pig atrium assay was that is, the uncertainty of toxin distribution or demonstrated (r = 0.88). Examination of non- localization in various parts of the tissue; (2) a toxic fishes and fishes recovered from patients tendency for false positive reactions due to non- with clinically defined ciguatera poisoning with specific binding; and (3) reproducibility due to the RIA procedure demonstrated that the test the fact that tissues are utilized, and even within clearly differentiated toxic from nontoxic sam- close proximity "hot spots" of ciguatoxin may ples. It is suggested that the RIA test in its vary. present state is useful for the assessment of toxic Efforts are currently being directed towards from nontoxic fish tissue in a variety of fish decreasing the test time, devising an ELISA pro- species from various endemic areas. Currently cedure using homogenous crude lipid extracts work is being carried out to develop a simpler from large tissue areas, minimizing the cost of enzyme-linked immunoabsorbent assay with the test, and increasing specificity by attempting increased specificity. to isolate and concentrate the specific anti- body involved. In addition, the development of immunofluorescent procedures wou Id be he1 p- LITERATURE CITED ful in assessing the presence of ciguatoxin in Bagnis, R. 1968. "Clinical aspects of ciguatera Garnbierdiscus toxicus (Figure 3) (Yasumoto et (fish poisoning) in French Polynesia." Hawaii al., 1977). Med. J. 28:25-28. The need for a specific, sensitive, and simpli- Banner, A.H., P.J. Scheuer, S. Sasaki, P: Helfrich, fied procedure for the detection of ciguatoxin is and C.B. Alender. 1960. "Observations on 6 I ciguatera-type toxin in fish." Ann. N. Y. Acad. February 1980. Scien, 90: 770-787. Okihiro, M.M., J.P. Keenan, and A.C. Ivy. 1965. Banner, A.H., S. Sasaki, P. Helfrich, C.B. Alender, " with cholinesterase and P.J. Scheuer. 1961. "Bioassay of ciguatera inhibition." Hawaii Med. J. 24:354-357. toxin." Nature 189:229-230. Oliver, G.C., Jr., B.M. Parker, D.L. Brasfield, and Becher, M. 1976. "Active groups in proteins and C.W. Parker. 1968. "The measurement of digi- their modification." In lrnmunoabsorbents in toxin in human serum by radioimmunoassay." Protein Purification, ed. E. Ruoslahti, pp. 11- J. Clin. Invest. 47: 1035-1042. 18. Baltimore: University Park Press. Rose, N.R., and H. Friedman, eds. 1976. Manual Butler, V.P., Jr., and S.M. Beiser. 1973. "Anti- of Clinical Immunology. Washington, D.C.: bodies to small molecules: biological and clin- Am. Soc. Microbiol. ical applications." Adv. lmmunol. 17:255-310. Scheuer, P.J., W. Takahashi, J. Tsutsumi, and Hokama, ,Y., A.H. Banner, and D. Boyland. 1977. T. Yoshida. 1967. "Ciguatoxin: isolation and "A radioimmunoassay for the detection of chemical nature." Science 155: 1267-1268. ciguatoxin." Toxicon. 15:317-325. Yasumoto, T., I. Nakajima, R. Bagnis, and R. Hokama, Y., L.H. Kimura, and T. Miyahara. Adachi. 1977. "Finding of a dinoflagellate as 1980. "Immunological approaches to under- a likely culprit of ciguatera." Bull. J. SOC.Sci. standing marine toxins." Paper presented at Fish. 43:1021-1026. the Sixth FDA Science Symposium, New Yasumoto, T., Y. Hashimoto, R. Bagnis, J.E. Orleans, La., 12-14 February 1980. Randall, and A.H. Banner. 1971. "Toxicity of Kosaki, T.I., B.J. Stephens, and H.H. Anderson. the surgeonfishes." Bull. J, SOC. Sci. Fish. 1968. "Marine toxins from the Pacific. Ill. 37 ~724-734. Comparative bioassay of ciguatoxin(s) in the mouse and chicken." Proc. West. Pharm. SOC. 2:126-128. AC K NOW LE DC EM E NTS Landsteiner, K. 1945. The Specificity of Serolog- This work was supported in part by the ical Reactions. Harvard University Press. United States Public Health Service; the Food and Miyahara, J., C.K. Akau, and T. Yasumoto. 1979. Drug Administration (RFP No. 223-79-22711; the "Effects of ciguatoxin and maitotoxin on the National Marine Fisheries Service (Honolulu isolated guinea pig atrium." Res. Comm. Laboratory); the United Fishing Agency, Ltd.; Chem. Path. Pharm. 25:177-180. the Honolulu Fish Dealers Association; the "Diseases acquired by inges- Morris, J.G. 1980. University of Hawaii Sea Grant College Program tion of fish and shellfish in the United States (Grant No. 04-8-MOI-178); and the Office of 1971-78." Paper presented at the Sixth FDA the State Marine Affairs Coordinator. Science Symposium, New Orleans, La., 12-14

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Sea Grant Quarterly (ISSN 0199-137x1 University of Hawaii second Clam Sea Grant College Program Portage Paid at 2540 Maile Way, Spalding 255 Hondulu, HI Honolulu, HI 96822 r-l