Toxfcat Vol . 29, No . 11, PP . 1387-1344, 1991 . Printed in Great Britain .

0041-0IOIJ91 53 .00+ .00 ~ 1991 Pergamoo Pros plc

AN ENZYME IMMUNOASSAY FOR THE DETECTION OF FLORIDA RED TIDE BREVETOXINS V. L. TYtAllvElt' and D. G. BADEN'~* 'University of Miami School of Medicine, Department of Biochemistry and Molecular Biology, P.O. Boz 016129, Miami, FL 33101; and =Rosenstiel School of Marine and Atmospheric Science, Division of Marine Biology and Fisheries, 4600 Rickenbacker Causeway, Miami, FL 33149, U.S .A . (Received 6 March 1991 ; accepted 7 May 1991) V. L. TRAINER and D. G. BADEN. An enzyme immunoassay for the detection of Florida red tide brevetoxins, Toxicon 29, 1387-1394, 1991 .-A noncompetitive solid-phase enzyme immunoassay for detection of brevetoxins in various matrices has been developed . The assay utilizes antibodies raised in a goat against brevetoxin PbTx-3-keyhole limpet hemocyanin conjugates with specific purification of brevetoxin antibodies through protein G and brevetoxin affinity columns, and rabbit anti-goat antibodies covalently linked to horseradish peroxidase . The assay was used specifically to detect brevetoxins in both cell culture and contaminated tissues. Sensitivity of the assay is 0.04 picomolar, and toxin can be quantified from 0.04 pM to 0.4 pM brevetoxin per well in microtiter plates by comparison with standard curves .

INTRODUCTION BREVETOX7NS are responsible for massive fish kills (BADEN, 1983), produce a smooth muscle depolarization in tracheal and bronchial tissues (RICHARDS et al., 1990; BADEN et al., 1982), and accumulate in bivalves during blood conditions (McFARREN et al., 1965). Incidences of undetected brevetoxins in coastal waters of the south-east U.S. have resulted in human illness and have recently been implicated in Atlantic Bottlenose Dolphin mortality (GERASI, 1989). The detrimental effect of algal blooms on public health enphasizes the need for an accurate and sensitive test for toxin detection. A principal difficulty in establishing the presence of brevetoxins, and in quantifying the amounts present, has been the lack of an analytical procedure possessing sufficient sensitivity . Analytical HPLC procedures exhibit detection limits of about 5 hg, and require extensive purification prior to analysis . Competitive radioimmunoassays (RIAs) with stated sensitivity of 2 nM have been available since 1984 (BADEN et al., 1984), and sensitivity has been improved by others to approximately 1 nM (PoLt, 1990a) . Although they are rapid, specific and reproducible, RIAs are not readily used under field conditions, due to the requirement for radioisotopes and expensive equipment for evaluation of results. "Author to whom correspondence should be addressed at: RSMAS/MHF, 4600 12ickenbacker Causeway, Miami, FL 33149, U.S.A . 1387

1388

V. L. TRAINER and D. G. BADEN HO OH

HO

KLH 75-100 :1

HO

FIG . 1 . BREVETOXIN PbTx-3 (TOP) WAS SUCCINYLATFA USING SUCCINIC ANHYDRIDE IN PYRIDINE AND WA3 THEN COVALENTLY COUPLED TO EITÜER KEYHOLE LIMPET HEMOCYANIN (MD)DLE) OR AMINOHEXYL SEPHARO3E HEADS (BOTTOM) USING STANDARD CARHODIIMIDE COUPLING PROCEDURES .

Coupling efficiencies of 75-100 umoles per imlole KLH and 1-3 ianmoles of toxin/ml AH Sepharose resin were achieved and efficiency was calculated using a small amount of tritiated PbTx-3 in the initial reaction mixture.

Enzyme-linked immunosorbant assays (ELISAs) have quantitative resolving capabilities (0.04-0.4 pM) which are several orders of magnitude higher than RIA (1 .6-43 nM) due to secondary antibody-enzyme conjugates which act as reporter molecules. In general, ELISAs are also more rapid than RIAs . With the expected advent of Federal guidelines for seafood testing in the near future, the brevetoxin ELISA described here could satisfy requirements for routine monitoring and quality control. MATERIALS AND METHODS Toxigenic organisms Prychodiscus brevis was originally obtained from the Department of Natural Resources in St Petersburg, Florida and has been maintained in our laboratories since 1973 . Gymnodinitan catenattan (Vigo strain) was purchased from the Toxic Dinoflagellate Culture Collection at Bigelow Laboratory for Ocean Science in Maine, U.S.A . Toxins Natural toxins were utilized as obtained . Brcvetoxins were purified from laboratory cultures of P. brevis (BADEN et al., 1981 ; PoLI et al., 1986). Synthetic tritiated PbTx-3 was produced by chemical reduction from

Brevctoxin ELISA

1389

PbTz-2 in the presence of cerium chloride and sodium borotritüde (Scour-st~ty et al., 1990). Homogeneity and quantitation of all toxin preparations was carried out using reverse phase C-18 high performance liquid chromatography (Ttt~uaett et a(., 1990). .lntigen

Hoth radioimmunoassays and enzyme-linked immunoassays utilize specific antibodies against brevetoxin PbTz-3 (Fig . l top). Total brcvetozin-bovine serum albumin (BSA) antigen construction has been reported previously (BADEN et aL, 1984). Coupling stoichiometries ranged from 10 to 13 .5 toxin molecules linked per BSA molecule . For the work reported herein, we synthesized a more immunogenic complete antigen, utilizing keyhole limpet hemocyanin as a carrier protein but employing the coupling procedure described previously. Coupling stoichiometries ranged from 75 to 100 toxin molecules bound per KLH molecule. Immwization serum manipulations

A single goat was immunized with the KLH conjugate on a biweekly interval with 0.33 mg toxin equivalents complete conjugate. The initial immunization was administered wiW complete Freund's adjuvant, and subsequent boosts were prepared with incomplete Freund's adjuvant . Ten bleeds were taken on alternate weeks for assessment of antibody titers using brevetoxin RIA (BenErr et al., 1984). Plasma from the resulting bleeds, and from larger scale plasmaphoreses were diluted with an equal volume of saturated neutral ammonium sulfate, placed at 4°C overnight, and centrifuged at 3000 x d for 30 min. The supernatant solution was retained and the pellet discarded. Addition of a second 50% volume of saturated ammonium sulfate to the supernatant solution (total volume twice initial serum volume) yielded an immunoglobulin precipitate which was separated from the supernatant solution by centrifugation at 5000 x d for 1 hr. The pellet was redissolved in 0.3 volumes of phosphate buffered saline (0 .l M, pH 7.4), dialyzed against PBS overnight, and frozen at -20°C. Protein G affably chromatography

A radial preparative protein G column (Genex GammaBind Plus, 50 ml bed volume, 1. ( g IgG binding capacity) was utilized in conjunction with a Bioltad 700 series High Resolution Liquid Chromatograph. Dialyzed samples were loaded on the column in waslting/binding buffer (0 .01 M sodium phosphate, 0.15 M sodium chloride, O.OI M EDTA, pH 7 .0) and the adsorbed IgG was washed with 10 column volumes of the same wash buffer . Adsorbed IgG was then eluted with 1 column volume of elution buffer (0.5 M acetic acid, pH 3 .0) and the elutee immediately neutralized with 1 M Tris base. Eluted IgG was dialyzed at 4°C against PBS overnight, lyophilized in 100ml aliquots, and stored at -20°C until further purified . Brevetoxin aj~nity chromatography

Brevetoxin PbTx-3 a(Snity columns were wnstructed using aminohexyl-Sepharose as solid support (l~lcmoles amino function/ml solid support; Pharmacia). Coupling of PbTx-3-succincte was achieved using standard carbodümide reactions with a trace amount (about 1 itCi) of tritiated PbTx-3 for assessment of coupling stoichiometry. PbTx-3 suociaate was synthesized using succinic anhydride as previously described (HADEN et al., 1984) . The derivative was purified by thin-layer chromatography and the succinic acid-toxin derivative was detected by spraying a portion of the developed plate wiW bromocrcsol purple . Aminohexyl-Sepharose (5 g) was washed with 3 x 50 ml of distilled water, followed by three 50 ml rinses of 50% pyridine . Five milliliters (~30 Etmoles of amino functional group equivalents) of the resulting slurry was added to 300lanoles of EDC (1-ethyl-3(-dimethylaminopropyl)carbodümide) in 0.5 ml of 50% pyridine. The mixture was shaken for 2 hr at room temperature, after which time 9.9 mg of PbTz-3-succincte (10 moles of toxin equivalents) was added in 1 ml of 50% pyridine . The reaction mixture was swirled at room temperature overnight, then packed in a 25 ml glass column, and rinsed sequentially with I column volume each of 50% pyridine, distilled water, and PBS, pH 7.4, without sodium azide. The resulting conjugate is shown in Fig. 1 (bottom) . The lyophilized protein G eluate was rehydrated and purified using the brevetoxin affinity column in the following manner. The column was washed with 10 mM Tris buffer, pH 7.5, followed by 10 bed volumes of 100 mM glycine (pH 2.5), 10 volumes of both 10 mM Tris (pH 8.8) and 10 mM Tris pH 7.5 . The pH of the column did not rise above pH 9.0, where brevetozins are known to be unstable (Pot.t, 1990b) . The sample was then loaded onto the brevetozin-affinity column in Tris buffer, pH 7.5 . Sample loading was accomplished most efficiently by recirculating the IgG through the column several times, evaluating specific binding by measuring protein in the elutee following each passage. Based on the calculated molar coupling efficiency of the column procedure, column binding capacity was approximately 1.6 g brevetoxin IgG. Following sample application, the column was washed with 20 column volumes of 10 mM Tris, pH 7.5, followed by elution, of the specifically bound brevetoxin antibody using 10 volumes of 0. l M glycine, pH 2.5 . Eluted IgG was neutralized with I M Tris base, dialyzed against PBS overnight, and stored in aliquots at -70°C.

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V. L. TRAINER and D. G. BADEN

F~c. 2. BxsvEroxnv ELISA PA01'000L. Serial dilutions of sample (100 pl) were added to 9Crrwell plates (Step 1) and allowed to equilibrate overnight at 4°C or for 2 hr at room temperature in PHS buffer. Unbound material was then aspirated from each well . Individual wells were rinsed three times with PBS, pH 7.2, then incubated with a blocking solution (1% non-fat dry milk in PBS, pH 7.2; HAat,ow and LArte, 1988) for 1 hr at room temperature (step 2). The solution was aspirated, followed by addition of brevetoxin antibody (serial dilutions ranging from 50 pg/well to 500 pg/well) and incubated for 1 hr in a humid chamber at room temperature (step 3) . The brevetoxin antibody was aspirated and retained for repurification, the plate washed with wash buffer, and rabbit anti-goat HRP conjugate (1 :1000 dilution) added to wells (step 4) . Following incubation for 1 hr at room temperature, the solution was aspirated, and wells were rinsed three times with wash buffer . Enzyme substrate (2 mM ABTS (2,2'-azino-bis-3~thylbenzthiazoline-6-sulfonic acid; Sigma Chemical Co., St . Louis, MO, U.S .A .) in 0.1 M sodium citrate buffer, pH 4.2, and 0.03% hydrogen peroxide) was added and changes in absorbance were monitored at 405 nm using a Hio-Tek ELr309 microtiter plate reader . ELISA Protocol

Dyaatech (Chantilly, VA, U.S.A .) Immulon~ 9Crwell microtiter plates were used for all studies. The optimized assay is depicted in Fig. 2. The protocol is a four-step procedure, beginning with sample adsorption to plastic plates, followed sequentially by nonspecific blocking of additional sites, specific binding of goat antibody to toxin, and finally, inwbation with rabbit anti-goat serum linked to horseradish peroxidase (Accurate Chemical Corp ., Westbury, NY, U.S .A.) . Comparative experiments were carried out using P. brevis and G. catenatam cells. Statistical analysis Using the statistical package Sigmaplot (Jandel Scientific, version 3 .1), correlations of cell concentration and optical density were made by regression analysis . RESULTS AND DISCUSSION

Immunization and titers

At a time when only the structures of PbTx-2 and PbTx-3 were known, we began developing radioimmunoassays for the detection of brevetoxins in marine food sources (BADEN et al., 1984). Utilizing BSA linked to brevetoxin PbTx-3 as complete antigen, we succeeded in producing antiserum in a goat. Characterization of immune sertun showed that the antibody had relatively low titer but high al$nity, with equilibrium dissociation constants calculated at 1 .32 nM. Subsequent work by PoLI (1990a) using identical

Brevetoxin ELISA

1391

TABLE I . PURIFICATION OF BREVETOXIN ANTIBODY

Protein

Activity* DPM/mg

Purificationfold

Preparation

mg

% yield

Serum Ammonium sulfate Protein G affinity Brevetoxin affinity

190

100

70

1

40

2l

552

8

24

13

4140

59

7 .2

3 .8

6822

97

*Activity is expressed as specific binding per unit protein at a fixed 2 nM [~H1PbTx-3 concentration . A total amount of 190 mg of serum protein was purified as described in Materials and Methods.

immunogen in different goats has illustrated the development of a higher titer of antibody with a similar affinity for toxin as demonstrated by us. Substitution of KLH-toxin for BSA-toxin in immunization procedures substantially increased antibody titers in serum, presumably due to the relatively insoluble nature of the KLH conjugate compared with BSA (ALLEN, 1985), the increased ntunber of toxin molecules bound per carrier molecule (Fig. 1, middle), and the higher molecular weight of KLH, factors all implicated in higher immunogenicity . Linkage of brevetoxin to the less soluble protein carrier did not appreciably alter the equilibrium dissociation constant of the antibodies elicited. Antibody pur~cation and characterization

Purification of brevetoxin antibodies from serum indicated that brevetoxin-specific IgG accounted for 3.8% of the total IgG population (Table 1). Purification of the brevetoxin antibody also reduced the nonspecific binding by first removing all potentially contaminating materials not of an IgG nature (using protein G chromatography) followed by removal of contaminating IgG not demonstrating a specific brevetoxin binding nature (using brevetoxin affinity chromatography). The purification scheme represented an approximately 100-fold increase in brevetoxin-binding activity per unit protein. The apparent equilibrium dissociation constant determined for crude antibody (1 .32 nM) did not differ appreciably from that determined for purified antibody (1 .30 nM). Non-specific binding was reduced to less than 10% of total binding by purification, in contrast to approximately 25% nonspecific binding exhibited by serum. SDS-polyacrylamide gel electrophoresis of samples at various stages of purification indicated that following protein G and brevetoxin affinity chromatography steps, all protein consisted of heavy and light gamma globulin chains of approximately 30,000 and 70,000 mol. wt . This further verifies the purification of antibody into its IgG components following the final chromatography steps. ELISA development

An important aspect of ELISA development is removal of non-brevetoxin antibodies which may interact with other reagents in the assay, e.g. specific antibodies elicited against the carrier protein during immunization . Nonspecific binding is observed in the inter-

a~ox ~9 :u-s

1392

V. L. TRAINER and D. G. BADEN

~o g .~ d 0

Aw 0 AO

0 .6

e

L) .O

Cell Concentration FIG .

3. ELISA USING

PROTEIN G-PURIFIED BREVETOXIN ANTIBODY INCUBATED WITH P. brevis (~) OR G. catenatum (O) CELLS.

Procedure is performed as described in text. Antibody concentration is 65 pg/well and incubation time is 2 hr . Average error of the mean of absorbance values demonstrating plate-to-plate variability is 6.1% (P. brevis) and 24.3% (G . catenatum) .

action of G. catenatum cells with crude brevetoxin antibody preparations which yields cell concentration-dependent color development (greater than 50% of the absorbance change observed with P. brevis at an identical cell concentration ; Fig. 3). Apparently, partially purified goat IgG contains non-brevetoxin antibodies which interact with dinoflagellate cell surface antigens . Affinity chromatography appears to remove most antibodies not demonstrating brevetoxin-specific recognition. Upon further purification using the brevetoxin affinity matrix, nonspecific antibody interaction with G. catenatum cells is reduced to less than 20% of the observed P. brevis cell response at the highest antibody concentration To>da Bquivnlents, aQ

Cell Concentration FIG .

4. ELISA

USING AFFINI7Y PURIFIED BREYETOXIN ANTIBODY INCUBATED WITH P. breY73 (~) OR G. CaterlatWR (O) CELLS .

Antibody concentration is 46 ~g/well and incubation time is 30 min. Average error of the mean of absorbance values demonstrating plate to plate variability is 6.9% (P. brevis) and 7.3% (G . catenatum) . Inset: Absorbance change over 1 hr at 1000 cell/well concentration.

Brevetoxin ELISA

1393

(Fig. 4). The ELISA response is linear with time, resulting in an overall absorbance change of 1 unit after 1 hr incubation with substrate (Fig. 4, inset) . HPLC quantification of toxin present on a per cell basis was correlated with the color development response in this ELISA . A linear correlation of absorbance change with cell number was observed at up to 100 cells per well after a 30 min incubation, allowing for direct quantification of toxin present in samples . It is appropriate that an assay be developed which detects brevetoxin associated with P. brevis cells, since toxin in shellfish is predominantly located within the gut tract as whole dinoflagellate cells and cell debris. Modification of this specific ELISA for use with toxin-contaminated tissue gave promising results . Ptychodiscus brevis cells were mixed with homogenized oyster flesh and subsequently tested for antibody response compared to cell controls. Although substantial nonspecific interaction was contributed by the oyster tissue, an increase in absorbance was observed with increasing P. brevis cell concentration after a 30 min incubation with the enzyme substrate . Future development of this assay for use in the field would require reduction of background nonspecific interaction . Alternative shellfish extraction protocols, different protein blocking agents, as well as more stringent washing conditions could be used to reduce nonspecific interference . Additionally, the assay could be modified into a format utilizing a convenient solid support system allowing for easy and precise toxin detection in field situations. In summary, assays employing affinity-purified brevetoxin antibody are far superior to any of those using protein G-purified antibody. The advantages to the assay are : (1) goat antibody is readily detected by a commercially available peroxidase-linked conjugate which can form multimeric complexes with its recognition sequences resulting in increased assay sensitivity ; (2) the ELISA design utilizes increasing toxin concentration in a direct protocol which results in increasing absorbance values . This allows for straightforward interpretation of results ; (3) average error of the mean of absorbance values demonstrating plate to plate variability is approximately 7% ; (4) the assay is linearly quantifiable from 0.04 pM to at least 0.4 pM brevetoxin per well . Federal guidelines mandate a legal limit of 80 ug of toxin per 100 g of shellfish tissue (20 mouse units/100 g; 4 ~g/mouse), currently analyzed by the traditional mouse bioassay. This ELISA shows several orders of magnitude greater sensitivity, illustrating its potential usefulness in monitoring seawater samples for early detection of coastal bloom conditions as well as in precise analysis of brevetoxin present in filter-feeding shellfish . Acknowledgements-This work was supported by U.S. Army Medical Research and Development Command under Contract No . DAMDl7-87-C-7001, and the Florida High Technology Council with Chiral Corporation acting as cost-share sponsor for toxin supply . Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the U.S . Army . In conducting research using animals, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH Publication No . 8023, Revised 1985 .

REFERENCES ALLEN, P. M., MCKe~N, D. J., BeCtc, B. N., $F~FF~L:LD, J. and Gr.tMC~x, L. H. (1985) Direct evidence that a class II molecule and a simple globular protein generate multiple determinants . J. Exp. Med. 162, 1264-1274 . BAUEN, D. G. (1983) Marine food-borne dinoflagellate toxins. Irtt . Rev . C'ytot. 82, 99-150. BADHN, D. G., McNUe, T. J., L~crr~rea, W. and WeLLHAM, L. (1981) Crystalliution and toxicology of T34: A major toxin from Florida's red tide organism (Ptvchodi.scus brevis). Toxicon 19, 455-462.

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BADEN, D. G., Meine, T. J., Ht1CHAZt, G. and LEUNC, I. (1982) Bronchoconstriction caused by Florida red tide toxins. Toxicon 20, 929-932. BADEN, D. G., BtLCtuzL, G., DectcEa, S. J., FOI.DES, F. F. and LEUNC, I. (1984) Neuromuscular blocking action of two brevetoxins from Florida's red-tide organism (Ptychodiscus brevis). Toxicon 22, 75-84. GFrust, J. R., ArroeLesoN, D. M ., TtntPeRt, R. J., Sr AuetN, D. J., EwLU.v, G. A., PRE4C01T, J. H. and Mwvo, C. A. (1988) Humpback whales (Megaptera novaengltae) fatally poisoned by dinoflagellate toxin. Can. J. Fish . Aq. Sci. 46, 1895-1898 . HAtu .ow, E. and LANE, D. (1988) Antibodies: A Laboratory Manual. New York : Cold Spring Harbor Lab. McFwLixetv, E. F., TANABE, H., Stt.vA, F. J., WILSON, W. B., CwtareeL.t., J. E. and Lewts, K. H. (1965) The occurrence of a ciguatera-like poison in oysters, clams, and Gymnodinium breve cultures. Toxicon 3, 111-123. PoL.t, M. A., MENDE, T. J . and BwneN, D. G. (1986) Brevetoxins, unique activators of voltage-sensitive sodium channels, bind to specific sites in rat brain synaptosomes . Mol. Pharmacol. 30, 129-135. Pot .t . M. A., TEEatrt,sroN, C. B., THOMP90N, W. L. and HewersoN, J . F . (1990a) Distribution and elimination of brevetoxin PbTx-3 in rats . Toxicon 28, 903-910. POLL, M. A., TEMPLfTI'ON, C. B., Pwce, J. G. and HtNfs, H. B. (19906) Detection, Metabolism and Pathophysiology of Brevetoxins . In : Marine Toxins : Origin, Structure and Molecular Pharmacology (HALL., S. and S~xtcxwterz, G., Eds) pp. 176-191. Washington, DC : American Chem . Soc. RLCxwßDS, I. S., KuLxwaNt, A. P., Bttootcs, S. M. and PIERCE, R. (1991) Florida red-tide toxins (brevetoxins) produce depolarization of airway smooth muscle . Toxicon 28, 1105-1112. SCHULINAN, L. S., R08ZELL, L. E., MENDe, T. l., KLNC, R. W. and BADEN, D. G. (1990) A new polyether toxin from Florida's red tide dinoflagellate Ptychodiscus brevis . In : Toxic Marine Phytoplankton (GRwxeLLa, E., SuNns~rROM, B., EnL.eR, L. and ArroexsoN, D. M., Eds) pp. 40712. New York : Elsevier Science. TRwtxeR, V. L., EDWARD3, R. A., $ZMANT, A. M., SMART, A. M., Meine, T. J. and BAneN, D. G. (1990) Brevetoxins: unique activators of voltage-sensitive sodium channels . In : Marine Toxins: Origin, Structure, and Molecular Pharmacology (HALL, S. and STRICHARTZ, G., Eds) pp. 166-175. Washington, DC: American Chem. Soc.