Todcar. Vol. 30, No. 7, PP. 66Wikl. Printed in Great hilain.

1992.

0041-0)101/92 55.w 0

+

1992pBpmoaRolLtd

m

MONOCLONAL ANTIBODY-BASED ENZYME-LINKED IMMUNOASSAYS FOR THE MEASUREMENT OF PALYTOXIN IN BIOLOGICAL SAMPLES GARY S. BIGNAMI,’ T. J. G. RAYIUXJLD,~~deceased,NAVZER D. SACHINVALA,* PAUL G.

GROTHAUS,~ SAMANTHAB. SIMPSON,CAROLYNB. Lao,’

JILANNEB. BYRNQ’ RICHARD E. Moor&’ and DOUGLAS C. VANN’

‘Hawaii BiotechnologyGroup, Inc., 99-193Aica HeightsDrive, Aica. HI 96701,U.S.A.; 2HawaiianSugar Planter’sAssociation.99493 Ake HeightsLhive. Aica, HI 96701,U.S.A.; and ‘Dqmrtmmt of Chemistry, Universityof Hawaii at Mmoa, Honolulu.HI 96822,U.S.A. (Rmiwd

18 November

1991;accepted6

Febntuty

1992)

T. J. G. RAYBWLD, N. D. SACHIIWALA,P. G. GRmU8, S. B. SWFSCIN, C. B. L.uo, J. B. BYRNFS, R. E. MGGRE and D. C. VANN. Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples. Toxicon 30, 687-700, 1!992.-Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-(ZV-maleimidomethylJcyclohexane-lcarboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palyrhoa tubercuZosu. The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enxyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosu extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection. G. S. BIGNAMI,

687

688

G. S. BIGNAMI rf al. INTRODUCI’ION

PALYTOIUN(PTX)* is a highly toxic natural product isolated from ‘soft corals’ of the genus Pulythou. The complete stmcture of PTX has been reported (mol. wt 26782681) (MOOREand BARTOLJNI,1981; UHMURAet al., 1981). Palytoxin is reputed to be the most potent non-protein animal toxin (HABERMANN,1989) and a potent non-phorbol ester-type tumor promoter (PUJIKI et al., 1986). PTX is also highly cytotoxic to mammalian cells in vitro (BONNARDet al., 1988). The potent and varied pharmacologic actions of PTX at the cellular, tissue, and organismal levels have been widely studied and are reviewed elsewhere (IBWIIM and &mu, 1987; d, 1989). Toxic Pdythou spp. have been identified in several of the world’s tropical and subtropical seas. The level of toxin in these soft corals may vary between species, between populations of the same species, and seasonally (Mooaa et al., 1982). KWUIU et al. (1973) reported increased toxicity of egg-bearing P. tuberculosu polyps. Others have suggested that PTX may be of bacterial (MOOREet al., 1982) or of algal origin (MAEDAet al., 1985), presumably existing symbiotically with Pulythou soft corals. PTX is reported to cause infrequent but severe, and even fatal, ciguatera-like seafood poisonings (YASUM~IDet al., 1986; FuKur et ul., 1987; ALCALAet al., 1988; KODAMA et al., 1989). Unlike ciguatoxin, however, PTX is water soluble and not likely to accumulate in fat tissue. Because PTX is less potent in mammals when administered by the oral route (Wnas et al., 1974) the reported acute intoxications suggest that tropical reef-dwelling fauna may at times consume sign&ant amounts of toxic Pulythou spp., or other PTX-containing organisms. HASHIM~~IIet al. (1969) found the remains of P. tuberculosu in the viscera of toxic 6legsh (Aluteru scripta). The potential effects of chronic exposure to low levels of PTX in the human diet are unknown, but may be of concern in view of the toxin’s reported potent tumor-promoting activity. A sensitive palytoxin-specific analytical method could be used to identify highly toxic Pulythou populations, to monitor PTX isolation procedures, and to quantitate the distribution of PTX in seafood comprising the human diet. Analysis of PTX-containing samples has been achieved by mouse bioassay (MOORE and SCHEUIR, 1971; TEEI and GNIDINER, 1974), by cytotoxicity assay (HBWEIWN et al., 1989; TAN and TEH, 1972) and by HPLC (UEMURAet al., 1985; YASVMO~,et al., 1986). Most recently, MEWED et al. (1991) have reported a highly sensitive high performance capillary electrophoresis detection method. In addition, LEVLNB et al. (1988) have developed a radioimmunoassay (RIA) incorporating [‘2511pTx. The RIA overcomes limitations of other methods for the assay of PTX in crude sample matrices by not requiring animals, a tissue culture facility, or extensive sample preparation. However, radioisotope use is potentially hazardous, usually

+Abbr&uions-AP, alknline phosphatawq AP-SH, thiolated alkal& phoaphatasc; AP-PTX. aikalhx phosphataae-palytoxin conjugal BSA, bovine serum albumiq CIEIA, competitive inhibition mymblinked immunoaawy; EIA, cnymblinkcd immunoassay; ELISA, enzyme-linked immuno+ent~asaay; CM, carboxymethyl; DEAE, dicthylaminocthy~ DMF,N&dimethylformami~ EDTA, ethylaedumme tctraacetic acid; KLH. keyhole limpet hcmocyanhq KLH-SH. thiolated. KLH; LF-MEM. kuciwfiee Eagle’s Minimal Essential Medium containing 10% calf serum. 1 mM sodium pyruvate. 2mM Lglutamiq PBS, 0.01 M sodium phosphate, pH 7.0.0.15 M Nac1; PBS-B, PBS containing 10 mg,/ml BSA; PBST, PBS contain@ 0.05% (v/v) Twccn-20; pNPP,pnitrophenyl phosphate, PT& pnlytoxiq PTX-MCC. N+I’~JV-malcimidomethyl)cyclohexanel’xarboxoyl)palytoxiin; P-IX-PDP. N-(3,-(2’-pyridyldito~onyl)palyto; BIA, radioimmunoassay; sulfc-SMCC. sulfoawinimidyl 4-(N-malcimidomcthyl) cyclohexanalsarboxylate; SPDP, succinhnidyl 3(2’-pyridyldithio)propionate; TBS-T, 0.05 M TripHCl, pH 7.0, 0.15 M N&l. 0.05% (v/v) Tween-20.

Enqmdinked

ImmmloMlays for Palytoxin

689

requires a licensed laboratory facility, and presents waste disposal problems. In addition, [‘zIJFTX is labile and must be prepared frequently for use in the RIA. Enzyme-linked immunoassay of PTX could retain the desirable features of RIA while eliminating the need for radioactive tracer preparation and use. Using defined P’IX haptens coupled to keyhole limpet hemocyanin (KLH), we have elicited PTX-specific rabbit polyclonal and murine monoclonal antibodies. These antibody preparations have been incorporated into competitive inhibition enzyme immunoassays (CIEIA) and into two-site sandwich enzyme immunoassays. These assays are useful for identifying toxic Palythoa and for monitoring toxin isolation. It is likely that they could also be used to survey seafood for PTX contamination. MATERIALS AND METHODS Chemicals Wacetylpalytoxin wan prepared according to the method of I-imkTAer d (1979). Citrate-free tetrodotoxin was purchased from Wochem (La Jolla. CA, U.S.A.). Okadaic acid was the girt of ALrONBOYTON. formerly of the Cancer Research Center of Hawaii, University of Hawaii. Lyngbyatoxin A wan isolated from &I&VI WI&W&J (CARD-A et al., 1979). Purity of these reagents WBB2 95% by ‘H-NMR. All other reagents were obtained from reputable suppliers. Palytoxin icomon FTX was isolated from Palythm tubercdoso c~llectal in Hawaii. Isolations were performed by the method of M~~RJZand SCHEUIB(1971). with modiftcations. PTX was extracted from P. t&rcuioso with 70% (v/v) aqueous

ethanol and concentrnted in vac~. The concentrated crude toxin preparation was &fatted by extraction with dichloromethane. PTX was puri!W from the combmed aqueous fractions by column chromatography on Amberlite XAD-2 (Sigma. St. Louis, MO, U.S.A.), followed by ion-ex&nge chromatography on DEAE-&phadcx A-25 (Sigma) and CM-Sephadex G25 (Sigma). Pm&d PlX was desalted on Bond Elut Cl8 (Analytichem International. Harbor City, CA, U.S.A.), elutal with 80% aqueous ethanol and lyophilized. Puritkd toxin ( > 95%) was &am&r&d by U.V. qe&oacopy (h = 23,600) (Moose, 1985) and ‘H-NMR. Standard solution of PTX were prepared in 50% aqueous ethanol and stored at -20°C.

of cru& extracts of P. tuhcrculoaa Crude extracts of P. tu&rcubsa (10 8) wm pqmcd by extraction with two volumes (w/v) of 70% aqueous ethanol for 18 to 24hr at room temperature. The extracts were centrifu@ and the decanted supematant was stored at -20°C. Over a @xi of we& precipitated material was observed in some of the extracts. When it was observed, the precipitate was removed by antrifugation prior to sampling. Preporot~

Hoptenprod&on

and conjugation to carrier proteins

Palytoxin haptens were prepared through selective derivatization of the primary amino functionality. N43-(2”-pyridyldithio)propimyl)pdytox~ (PTX-PDP). PTX was tnated with a tlvefold molar excess of the bifunctional linker SuaGnli dyl 3-Q’-pyridyldithio)propionate (SPDP) (Picroc. Rockford, IL. U.S.A.) in 0.1 M sodium phosphate butTer. pH 7.5:dimethyl sulfoxide @MSD)(l9:1) at 22°C for 4 hr. The reaction mixture was then partitioned between water and methylene chloride and extracted (3 x 0.5 ml CH$lJ to remove exaas linker. The aqueous phase was applied to a 1 ml Bond F&t Cl8 column (Analytichem International). Eluent from the Cl8 column was reapplied and the column washed with three column volumes of water. The crude product was eluted with 80% aqueo~ ethanol. The ethanol was sumoved in mcuo and the hapten preparation & further puriIied by ion-exchkge chromatography on CM-Scphadex C-25 equilibratal.wi& O.OiM-sodium oh-hate but&r. DH 4.5. Combined fractions containina muiikd hasten were desalted on a 1 ml Bond Elut Cl8 k&m, aa de&&d above. Following evaporation of‘ the e&ol, the hapten was diluted in water and lyophilized to yield puri6ed ITX-PDP. N_(~_(NI-thyl)~c~~~-c~~yl)~yto~ (PTX-MCC). PTX was treated with a fivefold dnol& excaa of the bit&ciional linker sulfos ua&&nidyl4-(Wmaleimido&hyl)cyclohexane&carboxylate 6mlfo-SMCC) (pierce) in 0.1 M sodium phowhate butTer.DH 7.S:dimethvl formamide (DMFW? 1) at 22°C for i hr. The ma&ion m&ture wa.3then pa&on-~ between titer and mcth&nc chloride ‘and &act& (3 x 0.5 ml CH,ClJ to remove excess linker. The aqueous phase was applied to a 1 ml kmd Elut Cl8 column (Analytichem International). Eluate from the Cl8 column was reapplied and the aAumn washed with three column volumes of water. The crude product was eluted with 80% aqueous ethanol. The ethanol was removed tn wcuo and the

G. S. BIGNAMI et al.

690

hapten preparation was fiuther purified by ion-change chromatography on CM-sephadex C-25 equilibrated with 0.02 M sodium phoephate bulTer, pH 4.5. Combinal fractions containing purified hapten wzre dcaalted on a 1 ml Cl8 Bond Elut wlumn aa described above. Following evaporation of the ethanol, the hapten was diluted in water and lyophilizcd to yield purifml P’IX-MCC.

haptens to kzyhok hpet hemoqah (kILlI) KLH was thiolatcd by stirring with a 5&fold molar excess of 2-iminothiolane in 25 mM borate buffer. pH 9.0, for lhr at 22’C. The thiolated KLH (KLH-SH) was subjected to buffer exchange on Scphadex G-25, euuilibratal with 0.1 M #odium phoS&ite. DH 6.6.1 mM EDTA. KLH-SH concentration was determined using BCA protein assayphe),*~0yh~mt3s Stanwhile free sulfhydryl groupe wcrc quantitated by themethodofG_ andh4~~(l%7).Themoleaoffreenulfhydrylpcrmole(1~g)KLH-SHwerethen calculated. P-R-PDP-KLH wan prepared by adapting the procedure described by W et al. (1978). Conjugates m dinlyzd against four chan8w of 0.1 M sodium phosphate buffer, pH 6.6.1 rnM EDTA. Protein and free eulfhydryl assays were repeated and the reduction in free aulmydryls per mole of KLH-SH was used to estimate the molar substitution ratio of KLH-SH with FIX-PDP, Conj+yationof &toxin

tie

is

Conjgation of paiytoxin haptens to BSA BSA WBBconiuaated to PTX-PDP m described above, for KLH conjugation. FIX-MCC-B!3A was prepared by stirring a &Told molar excess of PTX-MCC withBSA-SH (r&i& to net thiolation) for 1 hr at 22°C. Coniueatea were dial& asminst four channts of 0.1 M sodium phosphate buffer, pH 6.6,l mM EDTA. Protein and-f& sulfhydryl a&aya &re repeated a& the redtion in f&c su&dryls per-mole of BSA-SH was used to estimate the molar substitution ratio of BSA-SH with PTX-PDP or PTX-MCC.

of animals New Zealand White rabbits were given primary inoculations of 400 pg P-l-X-PDP-KLH in 1 ml PBS, pH 7.2, emulsiBed in 2 ml Freund’s complete adjuvant, and boo&d at 2-week intervals with 250~ of the same immw~ogen in Freund’s incomplete adjuvant. All injcc-tio~ were divided betwkn B.C.,i.m. and intradermal sites. Test bleeds were taken 7 days after each booster injection, and when indirect ELISA end point titers on these teat bleeds exceeded 1 in 106.the rabbits were sacriB& by Btion under general anesthetic. Female BALB/c mice wert given primary inoculationa containing 100 cc~ of PTX-PDP-KLH in 0.3ml Freund’s complete adjuvant, and boosted at Zweck intcrvah with 50~ of immunogen in Freund’a incomplete adjuvant. All injections were adminietcred i.p. Mice were bkd from the tail vein 7 daya &r each booster injection, to obtain sera for monitoring antibody titer against PTX-PDP-BSA. Mioz that were to be used 88 spleen cell donors for hybridoma production we= given 50 m of immunogcn i.p. in 0.2 ml PBS, pH 7.2,4 days prior to fusion. Im?nunisotion

Production of

nwnocti antibodies(mAb)

Hybrid0ma.s were w by polycthylcne glywl-maiiated fusion of splcnccytca from hypcrimn@zd BALB/c mice with the plammcytoma cell line. P3X63Ag8.653 (ATCC CRL 1580), according to the method of GODIN (1983). Hybridomas secreting the PTX-renctive mAbs were cloned three times by limiting dilution and cryoprtacmd in liquid nitrogen. Bulk quantities of mAb wzre harvested from the ascitic fluid of pristane+rimed BALB/c mice that had been inoculated i.p. with 2 x 106 hybridoma cells. The heavy and light chain isotype of mAba was determined by ELISA, using a commercially available kit (Zymcd. S. San Francisco, CA, U.S.A.).

Indirect EL&4

Indirect ELISA wan used for the determination of serum antibody titers and the screening of hybridomas. Immulon 2”’ microtiter plate wells (Dynatih Leboratorics, Chantilly, VA, U.S.A.) were coated for 1 hr at 22°C with 50 fl per wtll of PTX-MCC-BSA at an optimized concentration in PBS, pH 7.0. Plated wcrc wuhed three times with PBST. blocked for 1 hr at 22°C with 200 fi per well of 1% BSA (w/v) in PBS (PBSB), then washed again three timea with PBS-T. Fifty microliters of 8tflun or hybridoma culture rmpcmatant, titrated in PBS-B, wercaddcdto~wcll.andtheplatts~~~at220Cforlhr.AfltrwashingthreetimcainPBST.50Irlper well of goat anti-rabbit IgG and IgM s phosphatasc conjugate, appropriately diluted in PBS-B, were added, and the plates incubated at 22°C for 1 hr. Afta four linal washes in TBS-T, 200 ELI per well of 1 mg/ml pnitrophenyl phosphate @NPP)(Sigma) in AP sub&ate buffer (25 mM Trizma base, pH 9.5. 0.15 M NaCl, 5 mM MgCl, 0.02% [w/v] Na$) were added, and the absorbance of each well read after 60 min incubation at 22”C, on a Titertek Multiscan MC ELBA plate reader (sample wavekngth = 414 mn, reference wavelength = 690x11~)(ICN Plow, Costa Mesa, CA, U.S.A.).

Enxym~linked ImmuIloassays for Palytoxin

691

Indirect CIEIA

Indirect CIEIA was used to select mAba reactive with PTK in sohtion. Immulon 2’r” microtiter plate wells were coated for 1 hr at 22°C with 1004 per well of PTK-MCC-BSA at an optimixed concentrati& in PBS, pH 7.0. Plates were washed three times with PBST, blocked for 1 hr at room temperature with 200 4 per well of PBS-B, then washed again three times with PBS-T. Fifty microliters of puritied PTK standard or unknown sample, suitably dihrtcd in PBS-B, containing 0.5 mM Na,B,O,, and Sod of serum or hybridoma culture sunernatant. titrated in PBSB were added to each well. and the ~latca incubated at 22°C for 1 hr. Goat antimouse IgG + IgM alkahne phosphatase conjugate and enzyme s&&rate were prepared as described above. for the indirect ELISA, but 100~ volumes of the appropriate reagents were added to each well. Aiter four fmal washcsinTBST,#K)~~perwelloflmg/mlpNPPinAPeubstrate~~wereaddcd,andthcabeorbanaof each well read after 60 min incubation at 22”C, as described for the indirect ELISA. Antibody pur$calion Immunoglobulins were separated from rabbit serum by precipitation with 50% (v/v) saturated ammonium sulfate (Tusm~, 1985). Precipitated immunoglobulins wtrc stored at 4°C. Ammonium sulfate precipitated immunoglobulins were subjected to extensive dialysis against an appropriate buffer prior to use in assay development or isolation of IgG on Protein A-Sepharosee. An IgG,, rcmonoclonal antibody, 73D3, was selected for development of enxyme&ked immunoassays. The a5nity of mAb 73D3 for ti-MCC-BSA coating antigen was estimated by solid-phase ELISA to be 5.8 x 10s M-i (BIXT’IY et al.. 1987). Pol~clonal and mor&lonal antibodies w&e af&y puriiied from rabbit sera and mouse ascitic fluids by disco&nuous pH gradient elution from Protein A-Sepharoee@ (Protein A-Sepharose@ CL4B Data Sheet) (Pharmacia LKB, Piscataway, NJ, U.S.A.). Ehrted peaks were concentrated and buffer salts exchanged using CentriconTW 30 micrcconcentrators (Amicon. Beverly, MA, U.S.A.). Polyclonal and monoclontd antibcdiea were also ai%nity purified from rabbit sera and mouse ascitic fluids on Protein G-agarose (Gammabhrdru GAgarose, Genex, Gaitbersburg, MD), using the manufacturer’s directions. Preparation of antibody- and palytoximdhnlinephosphatawconjugates

AP was conjugated to rabbit anti-PTK IgG using a modiiication of the one-step gluteraldehyde method of Am (1969.1971). AP was treated with SMCC and conjugated to put&d mAb 73D3 previously thiolated using SAMSA. by the method of HXUKAWA et al. (1988). AP was thiolated for conjugation to PTK-MCC. AP was derivatixed with a 25fold molar excess of SPDP (dissolved in ethanol) in 0.1 M sodium phosphate buifer. pH 6.5. The reaction volume wax adjusted to 1 ml. keeping the iinal ethanol concentration below 1% (v/v), and the mixture was incubated with stirring at room temperature for 3Omin. The reaction mixture buffer was exchanged with 0.1 M sodium acetate but&r. pH4.5. using a GmtriconTM 30 microconcentrator. AP-PDP. in sodium acetate buffer, ph 4.5. was reduced by addition of DTT to a tinal concentration of 25 mM and incubated at 22°C for 38 min with stirring, to yield tbiolated AP (AI-!%-I). The AP-SH buffer solution was exchanged with 0.1 M sodium phosphate bu&r, pH 6.5, in a Centricon nt 30 microconcentrator. AI-SH was applied to a 1.0 ml Sepbadex G-25 spun column equilibrated with 0.1 M sodium phosphate buffer. pH 6.5, and centrifuged at 700 x g for 5 min (SAMFI~~XCI al.. 1989). Free aulfhydryl groups were determined, as described. The thiolated AP was reacted with a fivefold molar excess of PTX-MCC in 0.1 M sodium phosphate buBer, pH 6.5, for 14-18 hr at 4°C. The conjugate T tion (AP-PTX) was washed exhaustively with 0.1 M sodium phosphate butfer, pH 6.5, in a Centricon 343microconcentrator, in order to remove any unreacted PTK-MCC. l%zyme immwmways for PTX detection Indirect sandwich ELISA. Immulon 2TMmicrotiter plate wells were coated for I hr at 22C with 100 ~1 per well of Protein A-SepharoseQ purilied 73D3 mAb at an optimixed concentration in PBS, pH 7.0. plates were washed three times with PBS-T, blocked for 1 hr at room temperature with 200 fi per well of PBSB, pH 7.0. then washed again three times with PBS-T. One hundred microlhers of purified PTK standard or teat sample, appropria&ly dihrted in PBS-B containing 0.5 mM NarB,O,, were addedto each well, and the plates incubated at 22°C for I hr. Ah whim threetimes in PBS-T. 100 ul uer well of rabbit anti-PTX were added and incubated at 22°C for 1 br. A&r was&g three times in PBST, lob & per well of goat anti&tbbit 1%; ahtahne phosphatase conjugate, appropriately diluted in PBS-B, pH 7.0, were added, and the plates incubated at 22°C for 1 hr. After four final washes in TBST, 2004 per well of 1 mg/ml pNPP in AP substrate btier were added, and the absorbance of each well read after 60 min incubation at 22°C as described for the indirect ELISA. In the direct and indirect sandwich ELISA systems, PTK concentration in unknown samples was interpolated from the standard curve from the range (mean f S.D.) of absorbance at 414 nm that fell on the log-linear portion of the standard curve. These concentrations were then multiplied by the appropriate dilution factor, to give extract PTK concentration. D&ect sandwich ELIS.4. The dka3 sandwich ELISA method was performed as described for the indirect sandwich ELISA. except that AP-rabbit anti-PTX was used in place of rabbit anti-PTK. Consequently, the need for indirect detection with AI-goat anti-rabbit IgG was eliminated.

G. S. BIGNAMI CIal.

692 T-u

1.cQUPARJBDNOF

Solid phase coating reaecnt Pit soluble reagent Second soluble reagent Incubation timea Number of stepa+ Total amay time7 Aamy sensitivity at I% (xl/ml1 Asaay sensitivity at %I (nelml)

ENZYME-LJNKED IMMJUOASSAY SYSiENS FOR THE -N

Indirect sandwich ELISA

Op PN.Yl’CMN

Indirect CIEIA

Direu CIEIA with AP-73D3 hfAb

Direct CIEIA with AP-PTX

Dim3 sandwich ELISA

73D3 mAb

PTX-MCCBSA

PTX-MCC-BSA

73D3 mAb

73D3 mAb

Rabbit antiPTX AP-anti rabbit IgG 60min 7 >4hr O.a(l

73D3 mAb

AP-73D3 mAb

AP-PI-X

AP-anti mouse IgG 6omin 5 >3hr 6.2$

AP-Rabbit AntiPTX -

6Omin 3 2hr 3.q

6omin 3 :o.‘t;

35hr 4.811

0.37

1.69

0.68

3.1g

1.471

-

-

6omin

l Excluding coating and blwking, including washing. 7 Using previounly coated and blocked plater. tim required to obtain resulte. $ Palytoxin cowcluration required to produce 50% inhibition of @nal. 0 Palytoxin wnccntration required to produce 20% inhibition of rignal. IIm414 > 0.2 1 hr following addition of substrate. nori,,, > 0.1 1 hr following addition of substrate.

Indirect CIEIA u&g 7303. The idirad CIEIA mathod using 73D3 for PTX detection was identical to tbc indirect CIEIA methad described above for acmwing mAbs for maction with soluble PTX, except that optinudly diluted mouse aecitic guid wntaining 73D3 mAb was used in place of serum or hybridoma cultum eupanatant. ForthemeasurcmentofPI-XamndaK& and unknowns, SOpl of sample suitably diluted in PBSB wntaining 0.5 mM Na,B4G,. and SOpl of AP-73D3 conjugate appropriately diluted in PBS-B, were.added to each well, and the platen incubated at 22°C for 1 hr. Altar washing three timea in PBST, 100 pl per well goat anti-mouse IgG alkaline phoaphatam conjugate, appropriately diluted in PBS-B, pH 7.0, were added and tha platen incubated at 22”Cforlhr.Aftcrfourfiaal~~inTBST.200~1perwelloflmg/mlpNPPinAPsubetrateb~~were addad, and tha absorbance of each well read a&r 6Omin incubation at 22°C. w dencriwd for the indirect ELISA. In the CIEIA syotems, six control uninhibited wells were run in pamllel with each sample titration to provide a B, valua. A mean net abaorbana at 414mnfS.D., B, was cdcdati from the triplicate rem&a for each sample dilution. A mean B/B,, value was them calculated from them data. Sample dilutions with mean B/B, values falling on the log-linear portion of the standard curve were wed to interpolate 8ample PTX concentrations which were. than multinlied by tha anmomiate dilution factor. to give extract PTX wnccntration. Dtrect CIEIA wfng AP-73‘D3. ~~or1*2~ &titer plate welb~&wated for 1 hr at 22°C with 100 pl per well of PTX-MCC-BSA at an ontmnmd wncentration in PBS. uH7.0. Plates were washed three timea with PBST, blocked for 1 hr at room ~tiperature with 200 pl per well of PBS-B, then washed again three timer with PBST. Fifty microlitera of sample suitably diluted in PBS-B containing 0.5 mh4 NarB40,, and 50 pl of AP-73D3 conjugate appropriately diluted in PBSB, wcrc added to each wall, and the. plates incubated at 22°C for 1 hr. Afttrfourfinal~inTBST,200~clpaooflmg/mlpNPPinAPeubstratebuflcrwercadded,~the absorbanna of each well read alter 60 min incubation at 22°C. a8 demribed for the indirect ELISA. Dinct CIEIA wiqq AP-P7X. Immulon 2m microtiter plate welh were coated for 1 hr at 22°C with 100 rclper well of Protein A-S&ara&-puriflad 73D3 mAb at an- optimizad cwcwtration in PBS. pH 7.2. Plat& w& ~~~withPBST.Mockbdforlhratroom~~~with200ul~wcllofPBS-B.then washed again thrae times with PBS-T. FiBy microliters of sample mdtably dilutul h P-&-B wntaining 0.3 mh4 Na,B,O, and Sopl of AP-PTX conjugate appropriately diluted in PBS-B ware added to each well, and the plattsincubatcdat22’Cforlhr.Aftcrfour~waahcsinTBST,#W)~~pcrwellofI~mlpNPPinAP substrata buBer wera added, and the absorbance of each well read after 60 mitt incubation at 22°C. as described for the indimct ELISA.

Mouse bioauuy

Groupsof~female~Webster(~25g)mia~weiehcd,injcctsdi.p.withO.lmlofcodcd‘m~ unknown’ PTX samplea, diluted in 0.9%‘Naa’and tba time to death was monitored. Mean weights (kg) and

ATOX452P

00

MM26366

0412

MO34

Enzyme-linked Immml-ya

693

for Palytoxin

1.25 -+-

~bt

cm

wan)

FIG. 1. -AM STANDA~~D cullvm m PALYIOXM CIE!L4 -. cawementofPrxwcrcprcparedalJdcacribcdintheMa~and Standardcwveaforthcm Metboda. Each data point reprants mean B&&SD. for triplicate determinations.

1.0

0.9 48 0.7 0.6 0.5 0.4

~DiKa-EIxA

0.3

----t-hK6n!u-pLIsA

a2 0.1

o.or . .I

.--.--I

.

.

.

1

.----I

.

.

.

10

---..I

-

.

100

.-.---I loo0

~ytoxin~wm Ro. 2. Rnxmnrr~nw srmm CURVBS port PALYTOXM smmw-mi EL.ISA sysw~s. Standard curves for the measurement of FTX were prepared a9 described in the Material8 and Methods. Each data point rcprc=xntn mean net absorbance at 414nmfS.D. for triplicate dCMUliMtiOll8.

log dose palytoxin @g/kg) =

1.694

from linear rcgrcsioll (r’ = 0.849) of the sample data time points against the F-IX by U.V.qxcwscopy.

The c4plation was dcriwd

concentration dctcminal

log time to death @in)-0.601

G. S. BIGNAMI et a/.

694

Tm

Compound Palytoxin PTX-PDP+ N-Acetylp+toxin Heated-treated palytoxint 1 N HCypalytoti$ 1 N NaOH/palytoxid 10% Clorox@/palytoxinll Okadaic acid Tetrodotoxin Lyngbyatoxin A Guabain Bhamnose Amphotericin B Fiipin MoneMin NystatiIl

2. PALYTOXIN rmmo~~swcrprcrry(m~ Minimum detectable dose (ne/ml)

tcy) by w CIEIA @II/ml)

1 2

6 13 30 81 9 5 56 n.m.7 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m.

L

3i 3

3 10 > 1000 > loo0 > loo0 > 1.000.000 > 1.000.000 l.ooo.ooo 100,000 > 10,Oal > 10,am

CIEIA) BD~ by cytotoxicity assay @g/ml) n.d.:* 2OcKl > 1,ooo,OOo > l,OOO,am 5QO.ooo > 1.000.000 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

N-(r-(2”-pyridyldito~~onyl)palytox. 7 An aqueous solution of palytoxin maintained at 100°C for 12 hr. 3 Palytkin treated with 1N Ikl for 1 hr at room temperature. dpalvtoxin tnated with 1N NaOH for 1 hr at room temuerature. i Pa&toxin treated with 10% (v/v) Clorox for 1 hr at ro& temperature. 7 Not measurable at the hi&cat concentration taxted. l * Not determined. Indirect CIEIA and cytotoxicity aeeays were performed as described in the Materials and Methods. Each data point represents the mean of triplicate dctemkations. l

In vitro cytotoxicity aasayg were conducted with the murine T-lymphoma cell line, EL.4 (A TCC TIE 39). EL-4 cells were cultured at 37°C in Dulbuxo’s mod&d Eanlt’r medium (Whittaker Biomoducta. Walkemville. MD. U.S.A.) containing 10% calf serum (Hyclone, L.&n, UT. U.iA.), 1 mM &dium p&ate and i 4 L-glutamine under an humidikd atmosphere of 10% Co, in air. The assay wan conducted in leucin~frce Eagle’s Minimal Essential Medium (whittaker) containing 10% calf serum. 1mM sodium pyruvate. and 2mM L-glutamine (LF-MEM). Sam&s of p&led PTX w&e used to const&t standard cytot&city cuka. Purikd PTX and PTX unknown samples were diluted in LF-MEM and 504 aliquote were added in triplicate to the wells of sterile %-well microtiter plates (&star. Cambrids, MA. U.S.A.). Fifty microliter aliquots of EL-4 cells suspended in LF-MEM (2 x I@ cells/ml) were then added to each microtiter well. Samplea wzre gently agitated to mix the contentn of each well, and then the plates were incubated I5 hr under standard culture conditions. Vibility of the cuhurcs was determined by [“CJle tine incorporation. Each well was pulsed with 0.05&i [‘Qeucine in 5O/.d LF-MEM (DuPont New England Nuckar, Win, DE, U.S.A.; w activity 307.8 mCi/mmol) for 2 hr, prior to harvesting of the microtiter well contents onto glass fiber Alter disca. The tiltcrs were counted with a Beckman LS-7500 liquid scintillation counter. Data were expnscd a.s a mean percent [“C&u&e incorporation of treated culturea compared to untnated LF-MEM medium control cultures. Unknown sample PlX concentration wan determined by interpolation from the standard cum prepared with puriflcd PTX and multiplication by the appropriate dilution factor. RESULTS

Palytoxin isolation The isolation procedure described in the Materials and Methods section typically yielded 2.5-5 mg of purified PTX per kg (wet weight) of P. tuberculosu. Hapten production and conjugation to carrier proteins P’FX treated with SPDP produced PTX-PDP with Hal yields (based on starting PTX) of 60%. PTX treated with sulfo-MCC prcxhced PTX-MCC with final yields (based on

Enzyme-linked Immulloas&ly

0

for Palytoxin

10

5

695

15

al

palytoxin(cIBhnl) FKJ. 3. CQMPANSONoFINDrREcrCIEIAANDMouse BIOAS9AYKBRQLIANTTTATlONOFPALYTOXlN. Ebb mouse bioassay data point represents the mefm of five replicates. Each indirect CIEIA data point repmsenta tbe mean of triplicate dctcfminatio~. PTX standard solution concentration was d&t ’ cd by U.V. spectroscopy (+, = 23,600).

T~~re3.

Palytoxin concentration @./ml) Extract ll0.

1rnlinxsandwicl.1 ELBA meanfS.D. 2.9 0.3 3.9 2.4 11.5 0.9 0.4 25.6 49.9

% cv

'IIONIN P. h&rcuiosa EXTMCIS

~ATlONOPPALY-KlXlNcONCENI?lA

1.9 0.3 3.4 1.1 4.4 0.2 0.1 16.7 19.9 54

Each data point rcprwnta

Direct sandwich ELBA meanfS.D. 1.5 0.4 14.8 3.4 30.1 0.8 0.6 11.3 24.9

0.6 0.4 0.9 0.6 0 0 0 0.7 0 18

mcanfS.D.

calculated from results of:

Indimct CIEIA mcanfS.D.

Direct CIEIA with AP-73D3 mcanfS.D.

Direct CIEIA with AF-PTX mCUlfS.D.

3.2 2.5 28.4 5.7 22.9 1.2 0.3 18.1 24.3

2.6 1.7 6.6 2.1 9.0 0.9 0.3 25.9 22.2

3.0 3.5 20.7 4.7 22.9 1.4 0.2 11.0 13.4

0.2 0.6 10.3 0.5 3.3 0.1 0.1 2.6 4.6 17

0.3 0.6 0 0.8 1.1 0.2 0 9.1 12.5 27

0 0.4 1.6 0.8 0.9 0.1 0.1 4.2 0.9 15

for triplicate determinations.

starting PTX) of 54%. The structure of these haptens was confkmed by ‘H-NMR. (PTX-PDP: 6 8.1, amide proton; 6 8.5, 7.9, 7.4, pyridine ring protons) (PTX-MCC: 6 7.8, amide proton; 6 7.0, maleimide protons). Conjugation of PTX-PDP to KLH, for use as an immunogen, was optimized to yield w 8 moles of PTX-PDP per 10s g of KLH (0.1% [w/v] KLH, s21#)= 2.02 mgcm/ml) (GOOD et al., 1980). Substitution ratios were estimated by measuring the reduction in free thiol present in KLH following hapten conjugation. Conjugation of PTX-PDP to BSA, for use as ELISA plate coating antigen, was optimized to yield N 1.5-2.0 moles of PTX-MCC per mole of BSA. Substitution ratios were

G. S. BIGNAMI et al.

Ro.4.M-OP PALY~DXMEBXKIION~P. fubmdmun~~cI~ (AP-73D3). PTX was puriikd from P. ru&nxha (1~22kg) as daaibal in the Materiala and Methods. At theindicatcdstcp4sampleawcasti~~inPBS_Bandtcstcdintriplicataby~CIEIA,as described in the Materiala and Methods. PTX concentration of the diluted sampleswas interpolated from the log-linear portion of the standard curve and multiplied by the q&opriate dilution factor. The weight of PTX in isolation samplea was cnhxlated by multiplying the sample comrntration by the total volume of the isolation sample. For each sample, the value shown above mprescnta the average remultderived from two to three sample dilutions giving B/B, valueafalling on the log-linear portion of the aarldard curve.

estimated by measuring the reduction conjugation.

in free thiol present in BSA following hapten

Antibody production Polyclonal antibody was puri8ed from hyperimmune rabbit antiserum by precipitation with 50% ammonium sulfate followed by athnity chromatography using Protein A or Protein-G Sepharosem. Monoclonal antibody was harvested from the ascitic fluid of pristane-primed female BALB/c mice.

Characterization of alkaline phosphatase conjugates Anti-PTX rabbit polyclonal IgG-AP conjugate (approximately 2.8 mg/ml) had an endpoint titer of l/600 by direct ELISA on microtiter plates coated with PTX-MCC-BSA. The end-point titer is defined as the dilution of AP-conjugate required to produce an absorbance at 414nm of 0.2 units over background in the ELISA format. AP-73D3 conjugate (approximately 1.4 mg/ml) had an end-point titer of l/8000 by direct ELISA on microtiter plates coated with PTX-MCC-BSA. AP-MCC-PTX conjugates (approximately 1.4 mg/mI) prepared using both 1:1 and 5: 1 ratios (PTX-MCC to AP) were bound by 73D3 mAb adsorbed onto microtiter plate wells and exhibited high AP activity. However, the 5:l conjugate was useful at a greater dilution than the 1:l conjugate (l/2500 rather than l/500).

Ehzym~lillkd

Immunoassays for Palytoxin

697

EIA development A set of five EIA was developed for the detection of PTX. The required reagents, assay time, and assay sensitivities are compared in Table 1. Representative standard curves for the CIEIA and sandwich ELISA systems are shown in Figs 1 and 2, respectively. The direct CIEIA and direct sandwich ELISA depend upon conjugation of either PTX or antibody to alkaline phosphatase. We have found that stable APconjugates of PTX and both polyclonal and monoclonal anti-PTX antibodies prepared as described retain useful activity for more than a year, when stored at 4°C (data not shown). Specificity testing Each of the EIAs developed incorporated mAb 73D3. The cross-reactivity of 73D3 was therefore tested by use of the indirect CIEIA with various forms of altered PTX, other marine natural products, and certain ionophores, which might have relevance to the biological activity of PTX. The results of these experiments are shown in Table 2. PTX-PDP, N-acetylpalytoxin, and PTX treated with 1N HCl or 1N NaOH were serologically as reactive as native PTX. Heated PTX and Cloroxo-treated PTX were approximately l&fold less reactive than native PTX. Compounds chemically unrelated to PTX were at least looO_tirnes less cross-reactive than purified PTX. LEVINEet al. (1988) observed similar patterns of cross-reactivity with their polyclonal antibody-based RIA. The epitope recognized by 73D3 is preserved in certain PTX preparations that are virtually inactive pharmacologically, particularly 1N HCl or 1N NaOH-treated PTX. In contrast, treatment of PTX with heat or Clorox@, which also produces biologically inactive PTX, results in partial destruction of the 73D3 epitope. The cross-reactivity of 73D3 with N-acylated PTX suggests that the epitope recognized by this mAb is distal to the primary amino group of the toxin. Formation of a ‘double-antibody sandwich’ in the sandwich ELISA formats demonstrates that the PTX molecule contains at least two distinct epitopes. Comparison of indirect CIEIA and mouse bioassay The ability of PTX EIA to substitute for the mouse bioassay (MOOREand SCHEUER, 1971; TEH and GARDINER,1977) was evaluated with a series of coded samples containing varying amounts of purified PTX. Purified PTX concentration was measured by U.V. spectroscopy. As shown in Fig. 3, determination of PTX by indirect CIEIA agreed with the expected values at least as well as those obtained by the mouse bioassay method. Comparison of CIEIA and sandwich ELBA systems for detection of PTX in P. tuberculosa extracts Aqueous ethanolic extracts of P. tubercdosa were prepared as described in the Materials and Methods and assayed for PTX content by each of the five EIA systems. Table 3 shows the P’IX concentration (mean& S.D.) in nine crude ethanolic extracts determined separately by the five EIA systems, and the coefficient of variation for all the samples in each system. Spike and recovery The ability of an EIA system to accurately measure an analyte in a complex sample matrix can be assessed by spiking the samples with known amounts of purified analyte

698

G. S.

BIGNAMIer al.

and comparing assayed recovery with the predicted recovery (RODBARD,1981). Crude 70% aqueous ethanolic extracts were prepared from seven discrete colonies of P. tubercufosu, and spiked with an amount of PTX which would increase the PTX content of the diluted samples of each extract by 10 ng/ml. The untreated and spiked extracts were serially diluted and analyzed in triplicate by indirect CIEIA, using purified PTX to construct the standard curve. The indirect CIEIA was generally useful for detecting PTX in these sample extracts, as reflected in the overall percent recovery of added spike (mean = 103%). However, the recovery of spiked PTX was somewhat variable (S.D. = 29%), suggesting that several independent assays of an individual sample would be required to assure accurate PTX quantitation. Parallelism To test for possible matrix interference with the PTX EIA, crude 70% aqueous ethanolic extracts of P. tuberculosa were serially diluted in PBS-B containing 0.5 mM NqB,O, and analyzed by indirect sandwich ELISA, indirect CIEIA, and by both direct CIEIA systems. In the absence of matrix interference, the calculated extract PTX concentration should be equivalent when matrix dilution is accounted for. None of the CIEIA systems tested were subject to matrix interference. However, analysis by indirect sandwich ELISA revealed matrix interference with some, but not all, test extracts. In the sample showing the greatest degree of matrix interference, the calculated PTX concentration varied between 0.045 and 0.8 pg/ml ( w Is-fold), depending upon the dilution of the sample matrix. The source of the matrix interference was not defined. Four of the six extracts tested by indirect sandwich ELISA did not show any apparent matrix interference (data not shown). Monitoring of PTX isolation by direct CIEIA Three PTX isolations were monitored by direct CIEIA (AP-73D3), which indicated that approximately 30% of the toxin present in the crude 70% aqueous ethanolic extract was recovered as highly purified PTX. The results of these experiments are shown in Fig. 4. The greatest losses of PTX were incurred during the ion-exchange chromatography steps of the puri8cation procedure. DISCUSSION We have described the development of five EIA formats for the quantitation of F’TX in extracts of P. tuberculosa. As summarized in Table 1, each of the assays has certain attributes and disadvantages. The sandwich ELISA formats are capable of detecting PTX with excellent sensitivity, and do not require the use of palytoxin derivatives as coating antigen or tracer compound. However, the sandwich ELISAs may be subject to minor matrix interferences, as reflected in the non-parallel dilution phenomenon observed with some (but not all) crude P. tuberculosa extracts. The direct CIEIAs have the advantage of being rapid assays-when conducted with previously antigencoated and blocked plates, the assay of several samples can be performed in as little as 2 hr. The RIA reported by LEVINEet al. (1988) requires approximately 24 hr to perform. The sensitivity and specificity of these assays also compare well with the RIA reported by LEVINEet aC.(1988). The detection limits of the indirect and direct sandwich ELISAs are similar to the RIA, on a per test basis, but require a total test volume of only 100 fl,

Ellzymelinkcd Immwxlsnays

for Palytoxin

699

rather than 1 ml. The CIEIAs are slightly less sensitive, but do not appear to be subject to matrix interference. Each of the EIAs eliminates the need for [‘“IJPTX use. As observed with the RIA, the EIAs described here do not di scrimmate between native palytoxin and inactivated forms. However, no other serologically cross-reactive compounds were identified. Each of the EIAs should provide an adequate substitution for the mouse bioassay, as shown for the indirect CIEIA, in Fig. 3. The biological activity of critical samples can be congrnred by in vitro cytotoxicity assay, rather than by mouse bioassay (HEwarso~ et al., 1989). In general, we have found an excellent correlation between estimates of PTX content in crude and purilied isolates by EIA and in vitro cytotoxicity (data not shown). With purified PTX, assay by U.V. spectroscopy also correlates well with the other two methods. The EIAs described in this paper have been used to quantitate PTX in P. tubhcdosa extracts and to monitor toxin isolation. Using these EIAs, we have improved the average PTX yield per kg of wet raw material from approximately 1 mg/kg to as much as 5 mg/kg. Thus, the EIA have afforded improved efficiency of PTX isolation, promoting conservation of the P. tubercdosa resource. PTX was originally discovered in modem times by researchers seeking potential sources et al., 1969). of ciguatera seafood poisoning (MOORE and SCHEUW, 1971; H-ore Subsequently, the primary source of ciguatera poisoning was identified as ciguatoxin, from the dinollagellate, GumbierdLrcus toxicus (YMUMOTO et al., 1977). The possibility that FTX is responsible for at least some ciguatera-like poisonings remains (Yasu~oro and MURATA, 1990).KODAMAet al. (1989) reported RIA detection of palytoxin in extracts of mackerel (Decapterus mucrosoma) also testing positive for ciguatoxin. Because PTX is reportedly a potent tumor promoter (FIJJRG et al., 1986), chronic exposure to modest levels of the toxin could also present a human health hazard. The EIAs we have described are useful for identifying toxic PaZythoa spp. populations and could be used to survey fish and crab populations for the presence and concentration of FTX.

A&owle&emern.r-This work was supported in part by the U.S. Army Medical Research and Development Command, Contract Number DAMD17-87-C-7093. The technical assistance Of RAMONAVISNAK, J~~sN UNO and TOBUN CHEIINO. and the contract administration skills of DuNe Kuulz are gratefully acknowledged. This article is dedicated to our late colleague, Dr T. J. G. RAYBIXJLD.

REFERENCES

ALCAM, A. C.. ALCALA, L. C., GARTH, J. 8, YASUMUM, D. and Ym,

T. (1988) Human fatality due to ingestion of the crab Demmria reynuu& that contained a palytoxin-like toxin. Toxieon 26, 105407. Am S. (1969) Coupling of enzymes to proteins with gluteraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochembtry 6, 4352. A~MMEAS, S. and Tnrurv~ctc, T. (1971) Peroxidase labelled antibody and Fab conjugates with enhanced intracellular petration. Immvnochemisrry 8, 1175-l 179. BEA-rrv, J. D., BEAM, B. G. and VLAI-I~S, W. G. (1987) Measuremen t of moncclonal antibody affinity by noncompetitive enzyme immunoassay. J. Innarm. Met/f. lO& 173179. BCJNNARDC., Dx~una, J. F., Gnawr~. B. I., FIJJIKI,H. and HARRLq C. C. (1988) BBects of palytoxin or ouabain on growth and squamous ditTerentiation of human bronchial epithelial cells in vitro. Carcinogenesic 9. 22432249. CARD-A, J. H. II. Mm, F.-J. and Mooam, R. E. (1979) Seaweed dermatitis: structure of Lyngbyatoxin A. &&ace zor, 193195. CARLSON, J., Drmvrw, H. and AXEN, R. (1978) Protein thiolation and reversible protein-protein conjugation. N-sua5nimidyl 3-(2-pyridyldithiohxopionate, a new heterobifunctional linker. Biochem. J. 173. 723737.

G. S. BIGNAMI et ui.

700

FUJIKI, H., SUOANUMA,M.. NAKAYAW. M.. HUTI, H., HORIUCEII, T., TAKAYAMA, S. and Suonnrru, T. (19%)

Palytoxin ia a non-12-O-tctradacanoylphorbol-13-acttatc type tumor promotor in two-stage mouse akin carcinogeneaia. cor&ogene#i.v 7,707-710. FUKUI,M.. MUMTA, M., INCHJE, A., GAWEL,M. and Y,~MO’IQ T. (1987) Occumnce of pnlytoxin in the trigger 6sh Me&+ r&z. Toxicm 2!4, 1121-I 124. GoDuw, J .W. (1983) Production of monoclonal antibodies. In: MonoclorurlAn&x&s: principles mrdPraelicc, UD.56-97. London: Academic Press. G&D, A. H., Wopsy. L., HENRY,C. and KMJM, J. (1980) Reparation of hapten-modiiied protein antigens. In: Selecred A&h&s &I Cell&randImmww co bgy. pp. 343-350. (Mw B. B. and Smxx, S. M., Eds). San Francisco: W. H. Freeman groups with 2,2’- or 4,4’Grusserrr, D. R. and Mumuy, J.-F., JR (1967) Detem&ution of stiydryl dithiodiDyridinc.h&S &J&m. BiOUhYS.119, 41-49. I-&w&, E. (1989) Palytoxin acts &ugh tia+,K+-ATPaw. Toxicon 27, 1171-1187. w, Y., -ANI, N. and KIMURA,S. (1969) Aluterix a toxin of llle&h, Ah&t-a scripha, probably originating from a zoantharian. Palythoa h&rculoso. Bull. Jjm. Sot. scienk Fbh. 35,1086-1093. Hmwrso~, J. F., BI~AMI, G. S. and VANN,D. C. (1989) In vitro and in viva pro-on by monoclonal antibody against palytoxin exposwe. FASEB 1. 3, All91. HIUATA, Y., UEMUM, D., UI?D&K. and TAKANCI. S. (1979) Several compounds from Palyrhoa tu6ercubsa (Cudcnterata). Pure Appl. Ckm. 51, 1875-1883. IBMIUM,A.-R. and Swmx, W. T. (1987) Palytoxix me&minm of action of a potent marine toxin. J. Toxicol. Toxin Rev. 6, 159-187. ISHIKAWA. E., -A, S., KOHNO,T. and T~AKA, K. (1988) Methods for -labeling of antigens, antilxdica and their fragments. In: Nonisotopic Imrmmwua y, pp. 27-55. (Noo, T. T., Ed.). New York Plenum Press. KIMIn& s., HAwlwx0, Y. and YAMAZA ~1). K. (1973) Toxicity of the zaanthid Palyrhoa hdxmdosa. Toxicon 10,611417. KODAMA, A. M.. Houu, Y., Ym, T.. FUKUI,M., w. S. J. and Ss. N. (1989) clinical and laboratory findings implicating Palytoxin as cause of ciguatera poisoning due to Deerrplrrusmacrosmnu (Mackerel). Toxicon 27, 1051-1053. &~NE, L.. Furno, H., GJIKA,H. B. and VAN VUNAKIS,H. (1988) A radioimmunoassay for palytoxin. Toxicon 2&1115-1121. WA, M.. Kotuu, T., TANAKA,T.. Yowzu~~, H.. Nwm. K., Tn. T. and FUJXKI, T. (1985) Strwture of insecticidal sub&anaa isolated from a red alga. Chondria ammta. In: Sympathmr Papers, 27th Sympokm on Ihe Chemisrry of Nahual Praducrs, 6lti23; Chem. Absfr. 104,183,26Ot. m K. A., MORRIS, S.. Mw G.. TA~-LOR,T. J. and BUNNER,D. L. (1991) Analysis of palytoxin by liquid chromatography and capillary electrophoti. J. Llq. Chromu~. 14, 1025-1031. MOORE,R. E. (1985) Structwe of palytoxin. Prug. Ckem. org. nut. Prod. 48,82-202. MOORE,R. E. and BARTOLINI, G. (1981) Structure of palytoxin. J. Am. &em. Sot. 103.2491-2494. MOORE,R. E. and a, P. J. (1971) Palytoxinz a new marine toxin from a Coelenterate. Science 172, 495-498. Mmm, R. E.. Hmmum, P. and PAITERSCIN, G. M. L. (1982) The deadly seaweed of Hana. Oceauu U, 54-63.

ROD-, D. (1981) Mathematics and statistics of ligand assays: en illustrated guide. In: ugcmd Assoy, pp.45-102 WAN, J. and CUSP, J. J., Eda). New YorL: Maason Publishing U.S.A. SAMBROOK, J.. FRIZH, E. F. and MANIATIS, T. (1989) Spunxolumn chromatography. In: hfoleculor Cloning: a LmborororyMunuai, 2nd Edn, pp. M., Eds). Cold Spring __ E37-E38. (FORD.N.. NOLAN, C. and FFJ~OUSON, Harbor, h: Cold spring Harbor Laboratoj PTtBs. TAN, C. H. and m Y. F. (1972) The effect of Lo~zozpnw piclor toxin on HcLa cells. JZx@enlicr 28,46. TEH.Y. F. and GARDNER,J. E. (1974) Partial puri6cntion of Lqhozozymuspic~or toxin. Toxicon 12,603-610. Tug&, P. (1985) Purifk&on of &m&oglob& and preparati& of Fib f*ts. In: Luborafory Techniques in Biochem&wy and Mokcnhzr Biology, Vol. 15. Pracdceand Theoryof lkyme Immunoau ays. pp. 95-121 (BURWN, R H. end VAN -0, Eda). Amsterdam: Ehevier. Umeu~, D., USA, K. and HIRATA,Y. (1981) Further studies on palytoxin. II. Structwe of palytoxin. Tet. Len. 29.2781-2784. UJMUM, D., HIRATA,Y., IWASHITA,T. and NAOKI,H. (1985) Studies on palytoxina. Temhedrm 41,1007-1017. Wms, J. S., VICK,J. A. and w, M. K. (1974) Toxicologicnl evaluation of palytoxin in neveral animal ape&s. Toxicon Q427-433. YASUM~. T. and MURATA. M. (1990) Pol~~tlm toxina involved in seafood poisming. In: hfurirte Toxiru. Origin, &ruchwe, Mol&ar Piam&lo~. ACS Sympadum Serks. 418,-pp. 12&132 (HALL. S. and STRICHARTZ, G., Eds). Washin@on, DC: American Chemicnl Society. YASUMOTQ T., Yuuwu, M., ALCALA,A. C. and ti L. C. (1986) Palytoxin D., OHZUMI,Y.. Tm, in two species of xanthid aab from the Philippinea. Agric. Blol. Chem. SO, 163-167.

,BTOX452P

00

oooO22098 0511 ooO34