a monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples

Journal of Clinical Laboratory Analysis 6:65-72 (1992)

A Monoclonal Antibody-Based lmmunoassay for Detecting Tetrodotoxin in Biological Samples*

T.J.G. Raybould, G.S. Bignami, L.K. Inouye, Samantha B. Simpson, Jilanne B. Byrnes, RG. Grothaus, and D.C. Vann

Hawaii Biotechnology Group, Inc., Aiea, Hawaii

Spleen cells from mice hyperimmunized with a keyhole limpet hemocyanin-tetrodotoxin-formaldehyde conjugate were fused with murine P3X63Ag8.653 myeloma cells. A single hybridoma clone was identified that secretes an IgG,,k monoclonal antibody (MAb), designated T20G10, against tetrodotoxin ( T X ) , with an estimated affinityof 1.2 x lo* UM. Competitive inhibition enzyme immunoassays (CIEIAs) for detecting l T X were developed using this MAb. A direct ClElA using alkaline phosphatase-labeled MAb

detected mC with sensitivities at ICm and IC, of 6-7 ng/ml and 2-3 ng/ml, respectively. The accuracy of the direct ClElA was compara- ble with the high-performance liquid chromatography (HPLC) and the mouse bioassay systems, but the direct ClElA exhibited greater sensitivity. The direct ClElA was also more cost effective, as it required less sample preparation, a shorter assay time, and reduced investment in equipment than either of the other assay systems.

Key words: marine toxin, fugu, tetrodotoxin, monoclonal antibody, immunoassay

INTRODUCTION

Tetrodotoxin (TTX) is an extremely potent low-molecular- weight neurotoxin found in widely divergent animal species including puffer fish, gobies, salamanders, frogs, octopus, shellfish and starfish (1,2). TTX intoxication in humans most often results from ingestion of the flesh of certain species of puffer fish, in which elevated levels of TTX are found in the liver, ovaries and eggs. Raw puffer fish flesh, commonly referred to asfugu, is regarded as a delicacy in Japan and several other countries. Those who intentionally consume fugu seek a state of mild TTX intoxication, which is said to produce a pleasant peripheral “tingling” sensation. The raw flesh, however, may also contain TTX in sufficient concentration to produce severe intoxication, potentially leading to respira- tory paralysis and death.

The Japanese and U.S. governments currently require every puffer fish intended for human consumption to be tested. The standard method to assay for TTX is the mouse bioassay (3). This is an expensive and labor-intensive method that suffers from low sensitivity and the inability to discriminate among toxins. In spite of its greater sensitivity, an alternative bioassay employing the house fly (4) has not found wide accep- tance, because of the tediousness of such a test. An in vitro cell culture toxin neutralization assay system has recently been reported that employs a rabbit antiserum that cross reacts with TTX and saxitoxin (5). The sensitivity of this system for detecting TTX and saxitoxin was good (1 5 ng/ml TTX), but the assay was time consuming and did not discriminate between the two toxins. A number of HPLC methods have

0 1992 Wiley-Liss, Inc.

appeared in recent years (6-10). Each of these HPLC methods requires sophisticated equipment and time-consuming sample preparation procedures. In addition, the most sensitive of these HPLC methods was reported to have a detection limit of only 440 ng/ml of TTX (9). Bioreceptor assays using crude brain membranes can detect < I ng of TTX per milliliter of sample (1 1). The nature of these assays, however, precludes their application to large-scale screening procedures. Clearly, there is a need for an alternative, cost-efficient, test system.

Immunoassays are commonly used as rapid, inexpensive, sensitive, and highly selective methods for the detection and quantitation of a wide variety of drugs and other molecules of biomedical significance. Huot et al. (1 2) reported the production of two anti-TTX monoclonal antibodies (MAbs). These investigators used an immunogen prepared by treating a mixture of I T X and keyhole limpet hemocyanin (KLH) with an excess of formaldehyde. The resulting antibodies were screened against a bovine serum albumin (BSA) conjugate prepared in an analogous manner. A total of 9,329 hybridomas

Received August 30, 1991; accepted October 14, 1991.

Address reprint requests to Dr. T.J.G. Raybould, Hawaii Biotechnology Group, Inc., 99-193 Aiea Heights Drive, Aiea, HI 96701.

*In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, as promulgated by the Committee on the Care and Use of Laboratory Animals of the Insti- tute of Laboratory Animal Resources, National Research Council. The views of the authors do not purport to reflect the positions of the Department of the Army or the Department of Defense.

66 T.J.G. Raybould et al.

prepared from the spleens of immunized mice were examined to find two relevant clones. Binding of these MAbs was only partially inhibited (48% and 25%) by free TTX at a concentration of 50 ng/ml. Watabe et al. (13) immunized mice with a formaldehyde condensate of tetrodonic acid, a naturally oc- curring, nontoxic analog of TTX, and BSA. The resulting hybridomas were screened by an enzyme-linked immunosorbent assay (ELISA) system, using underivatized TTX as coating antigen. No indication is given of the amount of TTX that bound to ELISA plate wells but, in view of the hydrophilic character of TTX, it seems unlikely that significant concentrations of TTX would bind to a standard microtiter plate. Nevertheless, these investigators identified a MAb that detected TTX in this ELISA system at concentrations of 0.03-100 pg per well (0.3-1,000 pglml). Neither of these approaches has produced an immunoassay with the sensitivity required for determining TTX levels in puffer fish. However, they did indi- cate the potential of such an approach. We now report production of a hgh-affiity MAb against 'ITX and its use in developing sensitive and specific competitive inhibition enzyme immunoassays (CIEIAs) for detecting TTX in biological matrices.

MATERIALS AND METHODS

Tetrodotoxin

Citrate-free tetrodotoxin was obtained from Calbiochem.

KLH-TTXF Conjugate lmmunogen

Keyhole limpet hemocyanin-TTX-formaldehyde conjugate (KLH-TTXF) was prepared using a modification of the methods of Huot et al. (12) and of Chu and Fan (14); 300 pl TTX (1 mgiml), 700 pl 1 M sodium acetate buffer, pH 7.4, 12 1 ~1 KLH (34.9 mg/ml), and 60 ~ 1 3 7 % formaldehyde were added dropwise (in that order), to an amber glas vial while stimng on a vortex mixer. The reaction mixture was then placed on a shaker and incubated at 37°C for 3 days. After dialysis against four 1-L changes of 50 mM sodium phosphate, pH 7.0, containing 0.15 M NaCl (PBS) over 3 days at 4"C, the conjugate concentration was determined spectrophotometrically using an extinction coefficient at 280 nm of 2.02 mg*crdml(l5). KLH-TTXF conjugate for use as an immunogen was alum precipitated by addition of 800 pl 10% (w/v) AlK(S0&-12 H20 and approximately 500 p1 1 N NaOH, being careful to maintain the pH within the range of 3.5-8 during the pro- cess. The resulting precipitate was washed six times with 15 vol of ice-cold PBS by centrifugation.

BSA-lTXF Conjugate-Coating Antigen

Bovine serum albumin-TTX-formaldehyde conjugate (BSA-TTXF) was prepared using an adaptation of the method of Huot et a1 . ( 12); 700 p1 TTX ( 1 mg/ml), 300 pl 1 M sodium acetate buffer, pH 7.4, 179 pl BSA (at 33.6 mgiml), and 41 pl of 37% formaldehyde were added dropwise, in that order, to an amber glass vial, while stirring on vortex mixer. The

reaction mixture was then placed on a shaker and incubated at 37°C for 3 days. After dialysis against four 1-L changes of PBS over 3 days at 4"C, the conjugate concentration was determined spectrophotometrically using an extinction coefficient at 280 nm of 0.667 mg.cm/ml(lS).

Immunization of Mice

BALB/c female mice immunized with alum precipitated KLH-TTXF received a priming dose of 20-50 pg conjugate in complete Freund's adjuvant, administered to multiple subcutaneous sites, followed 1 week later by a subcutaneous booster immunization in incomplete Freund's adjuvant of 20-50 pg conjugate. Thereafter, these animals received weekly booster immunizations of 10-25 pg conjugate, administered intraperitoneally in PBS. Test bleeds from animals were monitored for appearance of a serum antibody response to BSA-TTXF. Those animals selected to be spleen donors for hybridoma production received daily intravenous boosts of 10-25 pg of conjugate in PBS on each of the 4 days prior to fusion.

Hybridoma Generation and MAb Antibody Production

Murine hybridomas were produced using standard procedures (16) with the fusion partner, P3X63Ag8.653. The resulting hybridomas were screened by indirect ELISA for antibody production against TTX. Hybridomas producing MAbs reactive with BSA-TTXF were tested for inhibition of this reactivity by 200 ng/ml unconjugated TTX by indirect CIEIA. Those cultures showing significant TTX-specific inhibition of solid-phase antibody binding were cloned by limiting dilution. Stable antibody producing subclones were expanded and cryopreserved in liquid nitrogen.

Primary ELISA Screen for Antibodies to TTX

Immulon 2 microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with 100 pl/well of BSA-TTXF at a predetermined concentration in PBS, for 1 h at room temperature. The optimum coating concentration for each batch of antigen was determined in advance. Typically, twice the minimum saturating concentration was used. Plates were then washed three times with PBS containing 0.05% (v/v) Tween-20 (PBS-T). After blocking with 200 pl/well of 1 % BSA in PBS for 1 h at room temperature, or 18 h at 4"C, microtiter plates were washed three times with PBS-T; 100 p1 of the sample being tested for anti-TTX antibody, titrated in 1% BSA in PBS, was added to each well, and plates were incubated at room temperature for 1 h. Microtiter plates were washed three times with PBS-T, then 100 FUwell of alkaline phosphatase- labeled goat antimouse IgG + IgM (H + L) conjugate (Caltag Laboratories, San Francisco, CA), appropriately diluted with 1% BSA in PBS was added. After incubation for 1 h at room temperature, plates were washed four times with 0.01M Tris- buffered 0.15M NaCl containing 0.02% NaN3 and 0.05%

MAb lmmunoassay for Tetrodotoxin 67

0.1 mlO.1 M Tris-HC1, and 0.1 ml hydroxylamine-HC1 was added and the mixture incubated for 4 rnin at 30°C. The thiolated T20G10 was transferred to a Centricon 30 (equilibrated with phosphate buffer, pH 6.0) and centrifuged with excess phosphate buffer for 20 min at 3,OOOg to remove unreacted SAMSA. The retentate was collected in a tared tube and tested by the DTDP assay (see below), to determine the molar ratio of sulfhydryl groups per antibody molecule. The thiolated T20G10 was mixed with afivefold molar excess of AP-MCC and stirred at 4°C for 18 h. The T20GlO-AP-MCC reaction mix was transferred to an Eppendorf tube and microcentrifuged for 5 min and the supernatant applied to a Sephadex G-200 column previously equilibrated with 10 mM Tris-HCl buffer, pH 6.8, containing 0.1 M NaCl, 1 mM MgC12, 0.1 mM ZnClz, and 0.5 g/L NaN3. The column was eluted using the same buffer and fractions collected and tested for AP-T20G10 activity by ELISA. ELISA-positive fractions were pooled, concentrated. and stored at 4°C.

(v/v) Tween-20 (TBS-T); 200 pl of 1 mg/ml p-nitrophenyl- phosphate (pNPP) (Sigma 104,5-mg tablets), diluted in alkaline phosphatase substrate buffer (25 mM Trizma base, pH 9.5, 0.15 M NaC1, 5 mM MgClZ, 0.02% (w/v) NaN3, pH 9.5) was then added to each well. The plates were incubated for I h at room temperature, and the absorbance of each well was read on a Titertek Multiskan MC using a sample wavelength of 414 nm and a reference wavelength of 690 nm.

Production of Ascitic Fluid

BALB/c mice were injected intraperitoneally with 0.5 ml of pristane (2,6,10,14-tetramethyl-pentadecane) (Sigma Chem- ical Co., St. Louis, MO) or incomplete Freund's adjuvant (IFA) (Sigmachemical Co., St. Louis, MO) (17). After 7-10 days in the case of pristane-primed mice, and 24 h in the case of IFA-primed mice, each animal was injected intraperitoneally with 5 X lo6 recloned T20G10 hybridoma cells. Mice were then observed for ascitic fluid accumulation. Ascitic fluid was withdrawn by an 18-gauge, 1.5-inch hypodermic nee- dle. Cells were removed from pooled ascitic fluid by centrifugation. The clarified fluid was then tested by indirect ELISA prior to being stored at - 20°C.

Affinity Purification of MAb From Ascitic Fluid

Affinity purification of murine MAb from ascitic fluid was performed by discontinuous pH gradient elution from Protein A-Sepharose (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) (18).

Alkaline Phosphatase-TPOGlO Conjugation

Alkaline phosphatase (AP) (Scripps Laboratories Inc., San Diego, CA) was conjugated to purified T20G10 MAb, using the method of Ishikawa et al. (19). AP was dialyzed against two changes of borate buffer (50 mM sodium borate, pH 7.6, containing 1 mM MgC12 and 0.1 mM ZnClz) at room temperature, then reacted with a 25-fold molar excess of sulfosuc- cinimidyl4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (sulfosuccinimidyl-MCC) (Pierce, Rockford, IL) in borate buffer for 30 min at 30°C; 100-p1 aliquots of the AP-MCC were applied to 1 ml Sephadex G-50 (fine) spun columns (20) that had been equilibrated with 0.1 M Tris-HC1, pH 7.0, containing 1 mM MgC12 and 0.1 mM ZnC12 (Buffer T), and centrifuged for 2 min. The AP-MCC filtrates were collected and concentrated on a Centricon 30 (Amicon Division, W.R. Grace and Co., Beverly, MA) that had been equilibrated with buffer T.

The protein A-purified T20G10 was concentrated and its buffer exchanged for 0.1 M sodium phosphate, pH 6.5 using a Centricon 30 (equilibrated with the same buffer). The protein concentration of the solution was estimated by measur- ing its ODzs0 using an extinction coefficient of 1.4 mg*cm/ml. The T20G10 MAb was thiolated by stirring with a 100-fold molar excess of S-acetylmercaptosuccinic anhydride (SAMSA) for 30 min at room temperature. Then, 0.02 mlO.1 M EDTA,

Determination of Sulfhydryl Groups (DTDP Assay)

mined using the method of Grassetti and Murray (21). The concentration of sulfhydryl groups in samples was deter-

Indirect ClElA for TTX Using T20G10 MAb

Immulon 2 microtiter plates were coated with 100 $/well of BSA-TTXF using the method described earlier for the primary indirect ELISA screen. Plates were then washed three times with PBS-T. After blocking with 200 pl/well of 1% BSA in PBS for 1 h at room temperature, or 18 h at 4"C, microtiter plates were washed three times with PBS-T; 50 p,l/well of the sample under test for the presence of free TTX, and 50 Fl/well of T20GlO anti-TTX murine MAb (at an optimal dilution previously established by titration) both diluted in 1% BSA in PBS, were added; the plates were incubated at room temperature for 1 h. The plates were washed three times with PBS-T; then 100 Fllwell of alkaline phosphatase-labeled goat antimouse IgG + IgM (H + L) conjugate, at an optimal dilution (previously established by titration) in 1% BSA in PBS was added. After incubation for 1 h at room temperature, the plates were washed four times with TBS-T; 200 $/well of 1 mg/ml pNPP, diluted in alkaline phosphatase substrate buffer, was then added. The plates were incubated for 1 h at room temperature and the absorbance of each well read on a Titertek Multiskan MC using a sample wavelength of 414 nm and a reference wavelength of 690 nm.

Direct ClElA for Tetrodotoxin Using AP-TPOGlO Conjugates

The method for the direct CIEIA was identical to that used for the indirect CIEIA shown above, with the following excep-


tions: (1) 50 ~l /wel l of AP-T2OG10 conjugate, at an optimal dilution in I % BSA in PBS (previously established by titration), was added to wells instead of 50 $/well of T20G10 anti-TTX murine MAb; and (2) the step involving addition of 100 p,l/well of alkaline phosphatase-labeled goat antimouse IgG + IgM (H + L) conjugate was deleted.

TABLE 1. Cloning Efficiency of Hybridoma T20G10

Cloning cycle no. Cloning efficiency"

1 213 1 2 1/89 3 30132 4 41147

asecreting clones/tested clones. Data Analvsis

actually produced ascitic fluid. Ten more BALB/c mice were

incomplete adjuvant (17), then injected intraperitoneally 24 106 recloned T20G10 hybridoma cells per

mice had produced ascites. This ascitic fluid had an end point titer of

104 in an indirect ELISA system using microtiter plates coated with BSA-TTXF and alkaline phosphatase- labeled goat antimouse IgG + IgM conjugate.

mg of this MAb was purified from 3.5

tion from Protein A-Sepharose (18). The specific activities

Purification of MAb by this method resulted in a 10- to 20-fold increase in ELISA specific activity, with a 13% yield of purified protein to total protein in ascitic fluid.

All samples tested by and were run in therefore by bmperitoned injection of0.5 Freund's licate, and the mean result of each set of replicates was calculated. BSA-coated wells treated with AP-conjugate and substrate were included on each plate to measure background color development. The mean OD414 of these wells was sub- tracted from the mean OD414 of each set of control and test replicates prior to data analysis.

The end-point titer of antibody preparations tested in the ELISA system was noted, together with the dilution that

addition. A working dilution of one-half this dilution was

CIEIA system. For indirect and direct CIEJAs, standard curves were con-

structed for each experiment using a set of TTX standards. B/Bo values for each standard curve were calculated by divid-

later with By 12 days after injection of cells,

in

Approximately in a net OD414 Of about 0.5 after substrate ml ofT20G10 ascitic fluid by discontinuous pH gradient elu-

employed for each antibody preparation in the indirect in ELISA of ascitic fluid and purified MAb were calculated.

ing the mean OD414 of a given set of replicates containing TTX inhibitor by the mean OD414 of all the wells containing no inhibitor. Unknown TTX concentrations in samples under test were calculated from the OD414 of the sample dilution(s) that fell within the linear portion of the standard curve. ICs0 and ICz0 values from standard curves were used to establish the sensitivity and cross-reactivity levels of the different CEIA systems.

RESULTS

MAb Production and Characterization

Two fusions were performed with spleen cells from the KLH-TTXF-hyperimmunized mice. The primary hybridomas from these two fusions were plated into 672- and 1,478- microtiter plate wells, of which 33 and 45, respectively, were positive in the primary ELISA screen for anti-TTX antibody.

Hybridoma T20G10 was isolated from the products of the first fusion. T20G10 was subcloned by limiting dilution four times; the cloning efficiency of this hybridoma line is sum- marized in Table 1. T20G10 produces an IgGl,k MAb. The affinity of this antibody has been estimated to be 1.2 x 10' L/M, using a solid phase ELISA method described by Beatty et al. (22).

Production of T20G10 Ascitic Fluid

Fourteen pristane-primed BALB/c mice were injected with hybridoma cells from clone T20G10. Only one of these mice

Indirect ClElA System Using T20G10 MAb

A 1: 1,000 dilution of the ascitic fluid detected free TTX at an ICs0 of 35-90 ng/ml and an ICzo of 12-30 ng/ml in a limited number of tests performed by indirect CIEIA system, using microtiter plates coated with BSA-TTXF and alkaline phosphatase-labeled goat antimouse IgG + IgM conjugate. Figure 1 shows the standard curve for T20G10 MAb in the indirect CIEIA system.

"1

0 8 -

BIB0 . a 06- Sensitivity IC50 = 90 ngiml IC20 = 30 nglml

1 0 -

0 8 -

06- Sensitivity IC50 = 90 ngiml IC20 = 30 nglml

lrrxl (nwW

Fig. 1. Standard curve for l T X detection by the indirect CIEIA. TTX was titrated in 1% BSA in PBS through the concentration range shown in the graph and samples tested, with controls containing no TTX, in triplicate by the indirect CIEIA. B/Bo values were calculated by dividing the mean of each set of replicates containing 'ITX inhibitor by the mean of all the wells containing no inhibitor, and plotted against TTX concentration. Error bars: % 1 SD.


Validation of Direct ClElA System

Reactivity with TTX-congeners The cross-reactivity of the direct CIEIA with other avail-

able TTX congeners was investigated. Tetrodonic acid was synthesized and purified in our laboratory according to the method of Tsuda et al. (23). This TTX congener did not cross- react in the direct CIEIA. In addition, samples of anhydrotetrodotoxin and tetrodonic acid were supplied to us by Dr. Takeshi Yasumoto of Tohoku University (Japan) and tested for cross-reactivity in the direct CIEIA. While the IC50 for TTX in this assay is 6-7 ng/ml, the ICso for these samples of anhydrotetrodotoxin and tetrodonic acid was 300 ng/ml and > 10,000 ng/ml, respectively. This indicates that the direct CIEIA is effectively TTX-specific.

Correlation of Direct ClElA System With the Mouse Bioassay and HPLC

The direct CIEIA was further validated by direct compari- son with the mouse bioassay and an HPLC method. The most sensitive HPLC assay for TTX is that reported by Yasumoto and Michishita (9) of Tohoku University in Japan. Thus, the HPLC measurements used for this study were performed by Dr. Yasumoto. The mouse bioassay was performed with Swiss Webster mice using the method of Mosher et al. (24), as described by Nachman (25).

A set of six TTX standards was prepared by serial dilution of a pure l T X solution, divided into three aliquots, and lyophilized. One aliquot of each standard was tested by direct CIEIA after reconsitution in 0.1 N acetic acid, another by mouse bioassay, and the third by HPLC analysis in Dr. Yasumoto’s laboratory.

In an attempt to provide further validation of the direct CEIA system without the availability of tetrodotoxic puffer fish, two large porcupine fish (Diodon hystrh), caught off Makapu’u Point, O’ahu, were obtained from O’ahu’s Sea Life Park. The porcupine fish is closely related to the puffer fish and has been reported to contain varying amounts of TTX (26). The livers were removed from these fish and samples prepared for analysis via the method of Yasumoto et al. (9) (Fig. 3). Since the toxicity of tetrodotoxic species is known to vary widely with the geographic location and few highly toxic examples are found in the reef areas of the Hawaiian islands (26) (Yasumoto T: personal communication, 1990), we could not be confident that these fish contained appreciable amounts of TTX. Therefore, both fish extracts were aliquoted; a known concentration of TTX was added to one-half of each sample, to produce a “spiked” sample. Spiked and nonspiked samples were divided into three aliquots and lyophilized. One aliquot of each sample was sent to Dr. Yasumoto for HPLC analysis. The second and third aliquots were tested by mouse bioassay and direct CIEIA, respectively. All samples were reconstituted in 0.1 N acetic acid prior to assay.

Table 2 summarizes the data from all three assays. The fish extracts to which TTX had been added (spiked samples) gave anomalous results in all three assays. The data from the TTX

Direct ClElA Using Alkaline Phosphatase-T2OGlO Conjugates

Modification of the indirect CIEIA to a direct system using AP-labeled T20G 10 antibody (AP-T20G 10) would provide an immunoassay requiring fewer reagents, fewer steps, and quicker test results than the unmodified indirect system.

Preparation of AP-T20G10 conjugates

Two alkaline phosphatase conjugates were prepared from Protein A-Sepharose purified T20G10 MAb using the thi- olating agent SAMSA and the heterobifunctional coupling agent sulfo-SMCC (19). Ratios of SMCC to AP of 25: 1 and 5:l were used with the aim of producing a conjugate with the best activity possible. A large amount of protein precipitation occurred in both preparations during the conjugation step, suggesting the formation of macromolecular aggregates. In spite of this precipitation, both conjugates exhibited acceptable levels of activity in ELISAs (reactive at dilutions down to 1:4,000- 1 :8,000, which represents conjugate protein concentrations of 0.27-0.53 Fg/ml).

Direct ClElA using AP-T20G10 conjugates

In the direct CIEIA system, each conjugate (at a working dilution of 1500, which represents a conjugate protein concentration of 4.26 pg/ml) reproducibly detected 5-10 ng/ml of tetrodotoxin. Thus, a direct CIEIA had been developed using an AP-MAb conjugate that exhibited sensitivity superior to the indirect CIEIA (that employs MAb followed by alkaline phosphatase-labeled goat antimouse IgG + IgM conjugate); the total test time was faster by 1 h (which represents a 30% reduction in total test time). Optimization of the direct CIEIA system enabled further increases in sensitivity and finally resulted in a TTX detection limit at an

of 2-3 ng/ml and an IC50 of 6-7 ng/ml (Fig. 2).

’“1 T

0.2 1 \

0.0 1 10 100 1000

IrrXl Wml)

Fig. 2. Standard curve for ‘ITX detection by the direct CIEIA using AP- T20G10 conjugate. mC was titrated in 1% BSA in PBS through the concenha- tion range shown in the graph and samples tested, with controls containing no TTX, in triplicate by the direct CIEIA. B/Bo values were calculated by dividing the mean of each set of replicates containing TTX inhibitor by the mean of all the wells containing no inhibitor, and plotted against ‘ITX concentration. Error bars: 2 1 SD.


interference from porcupine fish liver extract components and demonstrates that the time consuming sample preparation required for HPLC and mouse bioassay (see Fig. 3) is not necessary for the direct CIEIA.

Porcupine f i s h liver(10g)

1 . mamrate

3. oml to RT 4. Liler; wash paper with 25 ml 0.2 N HOAc

2. enran with 25 m10.2 N HOAc. 100 ‘C. 10 min.

P DISCUSSION I

Finrate I 5. enram wnh diethyi ether

Ether extract Aqueous extract (discard)

6. Amtsrlne CG-50, NH4+ (1.2 X 3 cm); a) wash wilh 50 ml water b) elute TI3 wirh 50 ml 0.5 N HOAc

7. adjust eluant10 pH 6 8. Charmal wlumn (1x5 cm) [desalting]

a) wash wdh 15 ml water b) wash wdh 25 ml 1 % HOAc c)elute TTX with 20% aq. ethanol

1 Eluant

I 1 Divide sample in han

a) spike wnh pure TIX b) divlde into thirds n c ) lyophilue

a) divide inlolhiids b) lyophilize

Unspiked Spiked samples samples ----

Assay by CIEIA, HPLC and mouse bioassay

1 rnl aliquots tested by ClElA spike and rewvev expenment

Fig. 3. Preparation of porcupine fish liver extracts for l T X determination.

standards were therefore plotted both with and without the data from the fish extracts (Figs. 4, 5) to establish the degree of correlation between the assays.

An aliquot of the porcupine fish liver crude extract fil- trate (see Fig. 3) and an aliquot of the defatted diethyl ether aqueous extract (see Fig. 3) were saved and tested by direct CIEIA. As TTX was not detected in either sample, a spike of 10 k g was added to each, and both retested by the direct CIEIA. The direct CIEIA detected approximately 10 kg in each spiked sample (data not shown). This observation indicates that the direct CIEIA was minimally affected by matrix

Undefined KLH-TTXF conjugates, prepared using the formaldehyde method of Huot et al. (1 1) were used as immu- nogens for stimulating high-titer antibody responses in mice. Two fusions using polyethylene glycol-mediated fusion of spleen cells from these mice with murine plasmacytoma cells resulted in the generation of large numbers of primary hybridomas. Based on Huot’s reports ( 12), however, we were not optimistic about finding many clones producing relevant antibodies. Repeated cloning by limiting dilution, of the 78 ELISA-positive wells from 2 150 original primary hybridomas finally resulted in the identification and isolation of one monoclonal hybridoma secreting a high affinity anti-?TX MAb (T20G10). MAb that had been affinity purified from ascitic fluid was used to develop a rapid, sensitive direct CIEIA for TTX detection and quantitation.

The direct CIEIA measures TTX concentrations in buffer or biological matrices with an ICz0 detection limit of 2-3 ngiml, which is significantly more sensitive than the immunoassays reported by Huot’s and Watabe’s groups (12,13). Excellent correlation for direct CIEIA, mouse bioassay, and HPLC was observed for a set of six TTX standards. This correlation was not as good when the porcupine fish liver extracts were assayed. Both the direct CIEIA and mouse bioassay over- estimated the amount of TTX “spike” that had been added to the extracts. This could be due to interfering substances in the matrix or, in the case of the mouse bioassay, greater sensitivity to TTX of the Swiss-Webster mice compared with the standard ddY mice (3). By contrast, the values determined by HPLC were far lower than the amount of TTX spike. Dr. Yasumoto reported that the lyophilized samples sent to his laboratory did not reconstitute satisfactorily. Incomplete dis- solution and/or decomposition of the TTX during shipment to Japan could explain these low values. Complete interpre-

TABLE 2. Correlation of TTX Direct CIEIA With Mouse Bioassay and HPLC’

[ITXI kg/ml as determined by Sample no. Weight Direct CIEIA Mouse bioassay HPLC

Standard 1 30.00 27.00 f 2.70 34.83 5 7.45 3 1.79 Standard 2 10.00 6.80 f 0.38 13.65 f 2.90 10.39 Standard 3 3.30 1.90 t 0.15 5.15 5 0.31 3.98 Standard 4 1.10 0.54 f 0.07 0.91 f 0.50 2.45 Standard 5 0.37 0.30 f 0.05 not tested 0.34 Standard 6 0.12 0.10 f 0.002 not tested 0.28 7 (fish 1) Unknown <0.008 nd <0GIb

9 (fish 2) Unknown <0.008 nd <O.Mb 8 (fish 1, spiked) Unknown + 25.00 40.32 f 2.82 30.92 f 5.14 16.96b

10 (fish 2, spiked) Unknown + 25.00 32.90 f 1.97 31.05 f 2.70 17.70b

“nd, not detected. brio other natural ‘ITX analogs were detected.


to screen for the presence of TTX in food for human consumption.

In this study, we have described development of a rapid, sensitive, direct CIEIA for TTX detection and quantitation. The accuracy of this immunoassay compares very favorably with existing assays, but is significantly more sensitive than the others. Finally, this immunoassay is considerably more cost-effective as it requires reduced sample preparation, assay time and investment in equipment than either HPLC or the mouse bioassay.

50 1 / /

Bioassay(stds) y - 0.33691 + 1.1699x R*2 - 0.994 0 HPLCWs) y - 0 4MM + 1 . W 7 x R^2 - 0.998

0 0 1 0 20 30 4 0 50

Fc] addad W m I )

Fig. 4. Correlation of direct CIEIA, HPLC, and mouse bioassay with l T X standards. A set of six TTX standards was prepared by serial dilution of a pure TTX solution, divided into three aliquots, and lyophilized. One aliquot of each standard was tested, after reconstitution in 0.1 N acetic acid, by direct CIEIA, another by the mouse bioassay, and the third by HPLC analysis in Professor Yasumoto's laboratory.

tation of this data is complicated by the fact that the porcupine fish contained no natural TTX. It is not clear whether the sample matrix produced from a toxin-negative fish is com- parable to that from a toxin-containing fish. It would have been preferable to have obtained naturally toxic fish from Japan for this experiment. This has not been possible as yet, however, because of logistical problems.

Although further testing will be necessary to confirm the utility of the immunoassay for determining TTX content of pufferfish tissues, we have demonstrated that minimal sample preparation is required for the direct CIEIA. In addition, it is preferable that the assay slightly overestimate rather than underestimate the TTX concentration if the assay is intended

1 /

1.1 / /

U

0 ClElA y I .O.67127 + 1.2285x R"2 - 0 901 Bioassay y - 0.20236 + 1.2065~ R"2 - 0.996 HPLC y - 0.41747+O.&X)OBr R12=0.926

0 1 0 2 0 30 4 0 50

mxl (wW

Fig. 5. Correlation of direct CIEIA, HPLC, and mouse bioassay with l T X standards and spiked fish extracts. A set of six TTX standards was prepared by serial dilution of a pure 'TTX solution, divided into three aliquots, and lyophilized. Extracts of the livers from two porcupine fish were spiked with 25 pglml of pure TTX, divided into three aliquots and lyophilized. One aliquot of each standard and lTX-spiked extract was tested, after reconstitution in 0.1 N acetic acid, by direct CIEIA, another by the mouse bioassay, and the third by HPLC analysis in Professor Yasumoto's laboratory.

ACKNOWLEDGMENTS

The authors acknowledge the cooperation of Dr. Takeshi Yasumoto and Dr. Mari Yotsu at the Faculty of Agriculture of Tohoku University, Japan, for their help and cooperation in this project. This work was supported by contract DAMD17- 87-C-7051 from the United States Army Medical Research and Development Command, Fort Detrick, Maryland.

REFERENCES

1. Mosher HS, Fuhrman FA: Occurrence and origin of tetrodotoxin. In Seafood Toxins EP Ragelis, ed. ACS Symposium Series, Vol. 262. Amer- ican Chemical Society, Washington, DC, 1984, p 333-344.

2. Bower DJ, Hart RJ, Matthews PA, Howden MEH: Nonprotein neuro- toxins. Clin Toxic01 18:813-863, 1981.

3. Kawabata T: Puffer toxin. In Food Hygiene Examination Manual, Vol. 11. Environmental Health Bureau, Tokyo, 1978, p 232-240.

4. Ross MR, Siger A, Abbott BC: The house fly: An acceptable subject for paralytic shellfish toxin bioassay. In Toxic DinoJlagellates DM Ander- son et al., eds. Elsevier, New York, 1985, p 433-438.

5 . Kaufman B, Wright DC, Ballou WP, Monheit D Protection against tetrodotoxin and saxitoxin intoxication by a cross-protective rabbit anti- tetrodotoxin antiserum. Toxicon 29:581-587, 1991.

6. Nakayama T, Terakawa S : A rapid purification procedure for tetrodotoxin derivatives by high-performance liquid chromatography. Anal Biochem 126: 153-155, 1982.

7. Bontemps J, Grandfils C, Dandrifosse G, Schoffeniels E: Reversed-phase separation of tetrodotoxin and its oxidized derivative at the C, ,-position detected spectrophotometrically at 195 nm. J High Resol Chromatogr Chromatogr Commun 7:265-267, 1984.

8 . Yotsu M, Endo A, Yasumoto T: An improved tetrodotoxin analyser. Agric Biol Chem 53:893-895, 1989.

9. Yasumoto T, Michishita T: Fluorometric determination of tetrodotoxin by high performance liquid chromatography. Agric Biol Chem 49:3077- 3080, 1985.

10. Onoue Y, Noguchi T, Hashimoto K: Tetrodotoxin determination methods. In Seafood Toxins EP Ragelis, ed. ACS Symposium Series, Vol. 262. American Chemical Society, Washington, DC: 1984, p 347-355.

11. Davio SR, Fontelo PA: A competitive displacement assay to detect saxitoxin and tetrodotoxin. AnalBiochem 141:199-204, 1984.

12. Huot RI, Armstrong DL, Chanh TC: Protection against nerve toxicity by monoclonal antibodies to the sodium channel blocker tetrodotoxin. JClinInvest 83:1821-1826, 1989.

3. Watabe S, Sato Y, Nakaya M, Hashimoto K, Enomoto A, Kaminogawa S , Yamauchi K: Monoclonal antibody raised against tetrodonic acid, a derivative of tetrodotoxin. Toxicon 27:265-268, 1989.

4. Chu FS, Fan TSL Indirect enzyme-linked immunosorbent assay for saxitoxin in shellfish. JAssoc OffAnal Chem 68: 13-16, 1985.

5 , Good AH, Wofsy L, Henry C , Kimura J: Preparation of hapten-modified

72 TAG. Raybould et al.

protein antigens. In Selected Methods in Cellular Immunology BB Mishell, SM Shiigi, eds. WH Freeman & Co., San Francisco, 1980, p

16. Goding JW: Production of monoclonal antibodies. In Monoclonal Anti- bodies: Principles and Practice. Academic Press, San Diego, 1983, p 56-89.

17. Mueller UW, Hawes CS, Jones WR: Monoclonal antibody production by hybridoma growth in Freund's adjuvant primed mice. J lmmunol Methods 87:194-196,1986.

18. Pharmacia LKB Biotechnology Inc: Protein A Sepharose CL-4B Data Sheet, Piscataway, New Jersey, 08854, 1988.

19. Ishikawa E, Hashida S, Kohno T, Tanaka K: Methods for enzyme- labeling of antigens, antibodies and their fragments. In Nonisotopic Immunoassay Ngo TT, ed. Plenum Press, New York, 1988, p 27-55,

20. Maniatis T, Fritsch FF, Sambrook J: Appendix A: Biochemical tech-

343-350.

niques. In Molecular Cloning. A Laboratory Manual. Cold Spring Har- bor Laboratory, 1982, p 466-467.

21. Grassetti DR, Murray JF: Determination of sulfhydryl groups with 2.2'- or 4,4'-dithiodipyridine. Arch Biochem Biophys 119:41-49, 1967.

22. Beatty JD, Beatty BG, Vlahos WG: Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Meth-

23. Tsuda K, Ikuma S, Kawamura M, Tachikawa R, Sakai K, Tamura C, Amakasu 0: Tetrodotoxin. VII. On the structures of tetrodotoxin and its derivatives. ChemPharm Bull 12:1357-1374, 1964.

24. Mosher HS, Fuhrman FA, Buchwald HD, Fischer HG: Tarichatoxin- tetrodotoxin: A potent neurotoxin. Science 144: 1100- 1 110, 1964.

25. Nachman RJ: Synthetic approaches to tetrodotoxin and analogs. Ph.D. thesis, Stanford University, 1981. cf. DissAbsInr41:4127-B, 1981.

26. Halstead BW: Poisonous and Venomous Marine Animals. Vol. 2. United States Government Printing Office, Washington DC, 1967, p 679-824.

ods 100:173-179, 1987.

a monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples

Documents