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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- fish industries. bodies to the haptenic molecules following This study discusses the immunological administration into appropriate animals. 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 fishes 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 Fisheries 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 Species 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 Seriola dumerilii Amberjackkahala 41 5,870 Toxic Seriola dumerilii Amberjack/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 Caranx 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.