Biol. Chem. Hoppe-Seyler Vol. 371, pp. 31-36, January 1990

Production and Characterization of Monoclonal Antibodies to Nivalenol Tetraacetate and Their Application to Enzyme-Linked Immunoassay of Nivalenol Hideharu lKEBUCHi a ,

, Kazuto HiRAi d , Michio SATOb,

Masakatsu ICHINOE C andTadao TERAO* a h c d

Division of Biochemistry and Immunochemistry Division of Medical Devices, and Division of Microbiology, National Institute of Hygienic Sciences, Tokyo Department of Hygienic and Public Health, Nippon Medical School, Tokyo

(Received 2 October 1989)

Summary: Three monoclonal antibodies were obtained by the fusion of mouse myeloma cells with splenocytes isolated from BALB/c mice that had been immunized with 8-hydroxy-3,4,7,15-tetraacetylnivalenol hemiglutarate covalently bound to bovine serum albumin. These anti-nivalenol tetraacetate monoclonal antibodies were of the IgG type and highly specific to nivalenol tetraacetate, with an apparent association constant of about 108 -1. The relative cross-reactivities of one monoclonal antibody with nivalenol tetraacetate, acetyl T-2 toxin, and scirpenol triacetate were found to be 1.0, 0.02 and 0.03, respectively. Other derivatives showed no cross-reactivity at all.

An indirect enzyme-linked immunosorbent assay (ELISA) based on the competitive binding principle was developed using the antibody from clone D18.102.59. The sensitivity of the system was about 0.1 ng of nivalenol tetraacetate per assay. Comparison of nivalenol levels detected in naturally contaminated barley samples by competitive indirect ELISA and gas chromatography (GC) showed good agreement, indicating that the antibody is useful for the measurement of nivalenol in naturally contaminated cereals and grains.

Bildung und Charakterisierung monoklonaler Antikörper gegen Nivalenoltetraacetat und ihre Anwendung für den Enzyme-linked immunoassay von Nivalenol Zusammenfassung: Durch die Fusion von MäuseMyelomzellen mit Milzzellen aus Balb/c-Mäusen, die vorher mit kovalent an Rinderserumalbumin gebundenem 8-Hydroxy-3,4,7,15-tetraacetylnivalenol-hemiglutarat immunisiert worden waren, wurden drei monoklonale Antikörper erhalten. Diese monoklonalen Anti-Nivalenoltetraacetat-Antikörper waren vom IgG-Typ und hochspezifisch gegenüber Nivalenoltetraacetat, mit einer apparenten Assoziationskonstante von etwa K^M"1. Die relativen

Kreuzreaktivitäten eines Antikörpers gegenüber Nivalenoltetraacetat, Acetyl-T-2-Toxin und Scirpenoltriacetat verhielten sich wie 1: 0.02: 0.03. Andere Derivate zeigten überhaupt keine Kreuzreaktivität. Mit Hilfe des Antikörpers aus dem Klon D18.102.59 wurde ein indirekter, auf kompetitiver Bindung beruhender ELISA entwickelt. Die Nachweisempfindlichkeit lag bei 0.1 ng Nivalenol pro Ansatz. Ein Vergleich der mittels kompetitivem indirektem ELISA erhaltenen mit gaschromatographischen Wer-

Abbreviations: Ac3 and Ac4 = tri- and tetraacetate, respectively; BS A = bovine serum albumin; GI-ELISA = competitive inhibition enzyme-linked immunosorbent assay; EDPC = l-ethyl-3-(3-dimethylaminopropyl)carbodiimide; ELISA = enzyme-linked immunosorbent assay; GC = gas chromatography; HG = hemiglutarate; HS = hemisuccinate; Ig = immunoglobuline; PBS = lOmM phosphate buffered saline, pH 7.2; RIA = radioimmunoassay; T2 toxin = 4/3,15-diacetoxy-3a-hydroxy-8a-(3-methylbutyryloxy)-12,13-epoxytrichotec-9-ene;TLC = thin-layer chromatography.

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32

H. Ikebuchi, R.Teshima, K. Hirai, M. Sato, M. Ichinoe andT.Terao

ten f r den Gehalt von Nivalenol an nat rlich kontaruinierten Gerstenproben zeigte gute bereinstimmung. Das zeigt, da der Antik rper f r die Messung

Vol. 371 (1990)

von Nivalenol an nat rlich kontaminiertem Getreide und anderen K rnern verwendet werden kann,

Key words: Monoclonal antibodies, nivalenol, nivalenol tetraacetate, trichothecenes, mycotoxins.

Nivalenol (3,4,7,15-tetrahydroxy-12,13-epoxytrichothec-9-en-8-one: Fig. 1) is a toxic metabolite produced by fungi of Fusarium species and often detected along with deoxynivalenol in food stuffs, especially in cereals and grains .lul Intensive research on the toxicity of nivalenol has been carried out13·41, and it has been pointed out that the acute toxicity of nivalenol in mice is stronger than that of deoxynivalenol.141 Although progress has been made in the development of analytical methods for trichothecene mycotoxins15'91, research on nivalenol is still hindered by the lack of a sensitive and rapid detection method. Assay systems useful for the measurement of mycotoxins include immunoassays with specific antibodies. Thus, radioimmunoassay and enzyme-linked immunosorbent assays employing polyclonal110'161 or monoclonal117'191 antibodies have been developed to detect trichothecene mycotoxins. Zhang et al. recently developed a radioimmunoassay system to measure deoxynivalenol.1141 In their study, they found that, although the presence of many hydroxy groups in deoxynivalenol has hindered the production of antibodies against this mycotoxin, once the hydroxy groups are acetylated, the modified molecule can serve as an effective immunogen after conjugation to protein.

o R« l CHa n3

Trichothecene Nivalenol Nivalenol tetraacetate Deoxynivalenol Deoxynivalenol triacetate Fusarenon X T-2 toxin AcetylT-2 toxin Scirpenol diacetate Scirpenol triacetate

Side -chain res due R4 R3

RI

*2

OH OAc OH OAc OH OH OAc OH OAc

OH OAc Η Η OAc OAc OAc OAc OAc

OH OAc OH OAc OH OAc OAc OAc OAc

OH OAc OH OAc OH Η Η Η Η

R5 =Ο =Ο =Ο =Ο =0 ISV ISV Η Η

Fig. I. Structure of nivalenol and related trichothecenes. OAc and ISVrepresent OCOCH3 and OCOCH2CH(CH3)2, respectively.

We found that the situation of nivalenol is similar to that of deoxynivalenol. Our attempts to produce useful antibodies against nivalenol in mice and rabbits after conjugation of this compound to proteins have been unsuccessful. Thus, we established a radioimmunoassay system using a rabbit polyclonal antibody to nivalenol tetraacetate (Teshima et al. unpublished work). In that study, we were unable to obtain satisfactory results by an enzyme-linked immunoassay system using rabbit polyclonal antibodies. This prompted us to produce monoclonal antibodies to nivalenol tetraacetate (Ac4nivalenol) for the development of an ELISA system. In this paper, we describe the preparation and characterization of mouse monoclonal anti-Ac4-nivalenol antibodies.

Materials and Methods Nivalenol, deoxynivalenol,T-2 toxin and scirpenol diacetate were purchased fromWako Chemical Co. (Tokyo, Japan). Nivalenol tetraacetate (Ac4-nivalenol), acetyl T-2, deoxynivalenol triacetate (Ac3-deoxynivalenol) and scirpenol triacetate were prepared as described previously'61. Bovine serum albumin (BSA, fraction V) and ovalbumin were purchased from Sigma Chemical Co. (St. Louis, MO.).Tri-rt-butylamine, isobutyl chlorocarbonate, glutaric anhydride and succinic anhydride were purchased from Wako Chemical Co. (Tokyo, Japan). Complete Freund's adjuvant was obtained from Difco Laboratories (Detroit, MI). All chemicals and organic solvents were reagent grade or better. Preparation of hemiglutarate of Ac4-nivalenol Hemiglutarate of Ac4-nivalenol (Ac4-8-OH-nivalenol-HG) was prepared as follows. Six mg of 8-hydroxy-tetraacetyl nivalenol (Ac4-8-OH-nivalenol), which was obtained by the reduction of Ac4nivalenol with sodium borohydride, was dissolved in 3 ml of dry pyridine with glutaric anhydride (48 mg) and N,W-dimethylaminopyridine (2 mg), and the solution was allowed to react for 4 h at 110 °C. After the reaction, distilled water (1 m/) was added to the reaction mixture. The aqueous solution was acidified with IM HC1 (pH 3) and extracted with 10 ml of chloroform. The organic phase was washed with 3% NaHCO3 solution three times (5 ml each), dried over anhydrous sodium sulfate and concentrated. Further purification of Ac4-8-OH-nivalenol-HG was achieved by preparative thin-layer chromatography (TLC) using a silica gel plate with chloroform/methanol (7:3) as a developing solvent. A silica gel band containing Ac4-8-OH-nivalenol-HG was scraped off from the TLC plate and extracted with chloroform containing 10% methanol to yield 3.5 mg of Ac4-8-OH-nivalenol-HG. GCmass spectral analysis after methylation of the compound with

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Vol. 371 (1990)

Monoclonal Antibodies to NivalenolTetraacetate

diazomethane showed a molecular ion of 610 (ΜΘ), consistent with the molecular mass of methyl-Ac4-8-OH-nivalenol-HG. Preparation of hemisuccinate ofAc^-nivalenol Succinylation of Ac4-8-OH-nivalenol was achieved by the reaction of Ac4-8-OH-nivalenol with succinic anhydride in the presence of N, /V-dimethylaminopyridine. Ac4-8-OH-nivalenol-hemisuccinate was purified as described above. Preparation of hapten-carrier conjugates Hapten-carrier conjugates were prepared by the mixed anhydride reaction with isobutylchlorocarbonate'20' or condensation with 1ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDPC). Briefly, Ac4-8-OH-nivalenol-HG (3 mg), tri-/7-butylamine (3.5 μg) and isobutylchlorocarbonate (1 μ/) in 100 μ/of dioxane were added to a 500 μ/ aqueous solution of BSA (10 mg/m/) and 1 μΐ of IM NaOH. The reaction proceeded overnight at 4 °C with stirring. After the reaction, the solution was loaded on a Sephadex G-25 column (1 x 15 cm) equilibrated with 10mM phosphated-buffered saline (PBS), pH 7.2. The conjugate was eluted with the same buffer, and fractions at the void volume were collected.This was used as immunogen. Ac4-nivalenol-hemisuccinate (Ac4-8-OH-nivalenol-HS) was coupled to ovalbumin by condensation of the hapten and ovalbumin with EDPC. The conjugate was also purified as described above. This was used as a solid-phase antigen in an ELISA. ELISA Ac4-8-OH-nivalenoI-HS-ovalbumin (50 μ/; 1 μg/m/in 0.15M carbonate buffer, pH 9.6) was added to each well of a 96-well polystyrene microtiter plate (Costar 96 well EIAplate, flat bottom, 3590) and incubated overnight at 4 °C.The wells were washed four times with 0.2 m/ of PBS containing 0.5% Tween 20 (PBS/Tween). For blocking of unoccupied solid phase-sites to minimize nonspecific binding, 200 μ/ of 0.1% casein in PBS was added and incubated for l h at 25 °C. The wells were washed four times with 200 μΐ of PBS/ Tween and then incubated for l h at 25 °C with 50 μ/ of culture supernatant (first incubation). The solution was removed by suctioning.The wells were washed as before and allowed to react for l h at 25 °C with 50 μ/ of a sheep anti-mouse IgG-/3-galactosidase conjugate (10~3 dilution, Amersham International Pic., Amersham, UK) in 0.1% casein in PBS (second incubation).The wells were washed four times with PBS/Tween and incubated for l h at 37 °C with 100 μ/ of O.lmM 4-methylumbelliferyl /3-D-galactoside (Sigma) in O.lM phosphate buffer (pH 7.2). Fluorescence was monitored byTitertek Fluoroscan. Competitive inhibition ELISA The procedure for competitive inhibition ELISA (CI-ELISA) was identical to that for the ELISA described above except that, in the first incubation, trichothecene as an inhibitor was simultaneously incubated for l h at 25 °C with a monoclonal antibody of an appropriate dilution with 0.1 % casein in PBS. The amount of bound antibody was determined as described above. Immunization Six 9-week old female BALB/c mice were immunized at 2-3 week intervals at multiple sites on the back with 0.2 ml of the immunogen, which was prepared by emulsifying 100 μg of Ac4-8-OHnivalenol-HG-BSA in 0.1 ml of sterilized PBS with 0.1 ml of Freund's complete adjuvant.Three mice which showed higher titers to Ac4-nivalenol were selected and received another intraperitoneal booster injection without the adjuvant.Three days later, spleen cells from these mice were collected.

33

Hybridization and cloning The immunized spleen cells and myeloma cells (NS-1) were mixed at a ratio of 3:1 and treated with 45% polyethylene glycol 4000 according to the method of Galfre et al .'21'. Fused cells were seeded in 24- well culture plates and selected in the HAT (hypoxanthine/ aminopterine/thymidine) medium. Antibody production of hybridomas was tested and then cloned by limiting dilution in the presence of thymocytes as the feeder layer. Antibody specificity and immunochemical characterization Antibody specificity was determined by the competitive inhibition of the ELISA as described above with various nivalenol-related trichothecenes as inhibitors and Ac4-8-OH-nivalenol-HS-coupled ovalbumin as the solid phase antigen. The Ig class, subclass and light chain of the antibodies were determined by the use of rabbit antibodies specific for mouse IgM, IgGb IgG2a, IgG2b or IgG3 and antibodies against mouse κ or λ light chain (10~5 dilution in PBS, Miles Laboratory, Elkhart, U.S.A.) as the second antibody in the ELISA. Radioimmunoassay (RIA) The competitive radioimmunoassay was performed as follows. One hundred μ/ of culture supernatant and 100 μ/ of [3H]Ac5-8-OHnivalenol (10000-13000 dpm) radiolabeled with [3H]sodium borohydride (8 Ci/mmol, NEN) were incubated with 100 μ/of various concentrations of Ac4-nivalenol at 4 °C overnight. After incubation , 300 μ/ of saturated ammonium sulf ate was added, and the mixture was kept standing at room temperature for 1 h. After centrifugation (3000 rpm, 20 min), the supernatant was discarded and the precipitate was dissolved in 300 μ/ of 10mM phosphate buffer. The radioactivity was measured with an Aloka model LSC-730 liquid scintillation spectrometer in 10 ml of Aquasol II (NEN).The association constant was calculated from the inhibition data according to Scatchard122'. Extraction and treatment of nivalenol from samples To test whether or not the monoclonal antibodies were applicable to the determination of nivalenol from cereals and grains, nivalenol was extracted from 10 g of contaminated barley with 30 ml of acetonitrile/water (7:3).The extract was then shaken with 30 ml of hexane and the acetonitrile/water layer was taken. The hexanetreated extract was evaporated to dryness. The residues were treated with acetic anhydride in pyridine for 2 h at room temperature to convert nivalenol to Ac4-nivalenol. Ac4-nivalenol was adsorbed to a C-18 cartridge (Waters Associates, Milford, MA) and the cartridge was washed with 50 ml of water. Ac4-nivalenol was eluted from the column with 9 ml of 50% aqueous methanol.The eluate was used for the CI-ELISA. Depending on the amount of nivalenol in samples, the eluates were appropriately diluted with O.lM phosphate buffer or phosphate buffer containing 10% methanol.

Results and Discussion Preparation of monoclonal antibodies Immunization of mice with nivalenol-BSA, which was prepared by coupling nivalenol-8-carboxymethyloxime to BSA raised antibodies specific to the nivalenol moiety of the antigen. However, all attempts to use these antibodies for RIA or competitive ELISA have been unsuccessful because the binding of nivalenol-protein conjugate and the antibodies was Brought to you by | University of Arizona Authenticated Download Date | 5/25/15 8:45 PM

34

H. Ikebuchi, R.Teshima, K. Hirai, M. Sato, M. Ichinoe andT.Terao 2 ^m

ble hybridoma clones. The supernatants were screened for the antibody production. The antibodypositive hybridoma cells were expanded and cloned. Only three clones were stable; these were designated as D18.102.59, J22.3.34, and L21.191.52.

o φ F 1

\ l Ο

Vol. 371 (1990)

2 4 6 8 Weeks after primary administration

10

Fig. 2. Production of antibodies against Ac4-nivalenol. Six 9-week old female BALB/c mice were immunized at 2-3 week intervals with Ac4-8-OH-nivalenol-HG-BSA and the increase in antibodies against Ac4-nivalenol was plotted. The arrows indicate each injection point. Antibody titer was defined as the reciprocal of the dilution of sera required to decrease binding by 50% under the ELISA condition described. The spleen cells of three mice (O—O) with high titers were used for hybridization. Error bars indicate standard deviation.

only very weakly inhibited by nivalenol (Ikebuchi et al., unpublished observation). On the other hand, once nivalenol was acetylated, antibodies against Ac4-nivalenol were readily observed. These results were similar to the finding by Zhang et al. for deoxynivalenol^14^ Therefore, we decided to use Ac4nivalenol as a hapten. The production of anti-Ac4-nivalenol antibodies in BALB/c mice immunized with Ac4-8-OH-nivalenolHG-BSA is shown in Fig. 2. Antibodies against Ac4nivalenol were detected at 5 weeks when the immunogen had been injected three times. High antibody titers were observed 9 weeks after immunization with our immunization protocol (five injections). The specificity of the antisera against Ac4-nivalenol and Acs-deoxynivalenol was determined by CI-ELISA. All the antisera reacted with Ac4-nivalenol and did not cross-reacted with Ac3-deoxynivalenol (data not shown). Therefore, the spleens of three mice which showed higher titers were selected and splenic lymphocytes were fused with NS-1 cells. Among 288 cultures seeded with fused cells, 158 (55%) yielded via-

Characterization of the monoclonal antibodies Ig-classes and -subclasses of the monoclonal antibodies were determined. The antibodies from clones D18.102.59, J22.3.34, and L21.191.52 were of Igd; κ, IgGi; κ, and IgGi; λ types, respectively. Association constants of these antibodies were 1.90 x 108, 1.28 χ ΙΟ8, 8.0 x 107M~!, respectively, as calculated by Scatchard analysis after the radioimmunoassay. The binding inhibition curve of the monoclonal antibody from clone D18.102.59 produced by Ac4nivalenol is shown in Fig. 3. Inhibition was nearly linear between 0.5 and 50 ng/m/ (Fig. 3).The concentration of Ac4-nivalenol causing 10 to 15% inhibition of binding was around 0.1 to 0.5 ng/m/. Since the standard deviation in the assays was normally within this range, it is reasonable to assume that the lower limit for the detection of Ac4-nivalenol under the present CI-ELISA conditions also falls within this range. Although this detection limit is somewhat higher than that for anti-Ac4-nivalenol antisera obtained in rabbits in our laboratory, it is sufficient for most analytical purposes. The specificities of these anti-Ac4-nivalenol monoclonal antibodies to several related trichothecenes were tested in the CI-ELISA system (Table 1). These antibodies did not cross-react with nivalenol, deoxynivalenol, Ac3-deoxynivalenol, T-2 toxin, or

2000

MOOO

1

10

100

50CO

Nivalenol tetraacetate [ng/m/| Fig. 3. Competitive indirect ELISA curve for monoclonal antiAc4-nivalenol antibody (clone D18.102.59) with various concentrations of Ac4-nivalenol as an inhibitor. Culture supernatant at a dilution of 1:450 was used as the antibody. Solid phase antigen was Ac4-8-OH-nivalenol-HS-ovalbumin. Error bars indicate standard deviation.

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35

Monoclonal Antibodies to NivalenolTetraacetate

Vol. 371 (1990)

Table 1. Specificities of monoclonal anti-Ac4-nivalenol antibodies. Trichothecene

Antibodies D18.102.59

Tetraacetyl-8-hydroxynivalenolhemiglutarate Tetraacetyl-8-hydroxynivalenolhemisuccinate Nivalenol Nivalenol tetraacetate Deoxynivalenol Deoxynivalenol triacetate Fusarenon X T-2 toxin AcetylT-2 toxin Scirpenol diacetate Scirpenol triacetate a 3

a

2.3 3.0 > 1,000 3.6 > 1,000 > 1,000 > 1,000 > 1,000 180 > 1,000 119

J22.3.34 b

(1.6) (1.2) (< 0.001) (1.0) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (0.02) (< 0.001) (0.03)

L21.191.52

(2.5) (1.4) (< 0.001) (1.0) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (0.16) (< 0.001) (0.23)

2.1 3.7 > 1,000 5.2 > 1,000 > 1,000 > 1,000 > 1,000 33 > 1,000 23

(2.2) (1.2) (< 0.001) (1.0) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (0.2) (< 0.001) (0.04)

5.0 9.2 > 1,000 11 > 1,000 > 1,000 > 1,000 > 1,000 56 > 1,000 278

The concentration (/xg/1) required to give 50% inhibition of the antibody binding in CI-ELISA. concentration of nivalenol tetraacetate Expressed as relative cross-reactivity ( causing50% inhibition). concentration of trichothecene

scirpenol diacetate at a concentration of at least 10 The fact that these antibodies did not cross-react with Acs-deoxynivalenol indicates that the acetoxy moiety at the C4 position plays an important role in recognition by the antibodies. Since Ac4-8-OH-nivalenolHG and Ac4-8-OH-nivalenol-HS reacted much better than Ac4-nivalenol, the keto group at the C8 position is not essential for recognition by these antibodies. Therefore, it is conceivable that the acetoxy group at the C7 position is also essential for the recognition since acetyl T-2 toxin was only weakly cross-reactive to the antibodies. Accumulated data have revealed that nivalenol was detected along with deoxynivalenol from cereal products in Asian^1'23'25' and European countries^24'26lThe monoclonal antibodies in the present study can discriminate Ac4-nivalenol from Aca-deoxynivalenol. Therefore, these antibodies are expected to be effective for the determination of nivalenol in cereals and grains, regardless of co-contamination with deoxynivalenol. Recovery of nivalenol added to barley samples The effectiveness of the ELISA procedure for the detection of nivalenol in barley samples was tested by adding different amounts of nivalenol to the blank barley extracts (acetonitrile/water extract whose nivalenol content was less than the detection limit of CI-ELISA). Nivalenol-spiked barley extracts underwent hexane extraction, acetylation, cartridge treatment, and CI-ELISA. Results are given in Table 2. For a sample spiked with a small amount of nivalenol (33 ppb), the recovery was more than theoretical (124.2%). In contract, for samples spiked with a large amount of the compound (670 and 3300 ppb), the re-

covery was less than the theoretical value (71.5 and 69.7%, respectively). Analysis of naturally contaminated samples In a further attempt to test the effectiveness of CIELISA of nivalenol in naturally contaminated samples, 6 barely samples that had been analysed for Table 2. Recovery of nivalenol (NIV) from spiked barley extracts by competitive inhibition ELISA. Each sample extract was spiked separately and pretreated according to the protocols in this study.

a b

NIVadded ppb

η

NIVdetected by ELISA ppba

Recovery [%] (±SD)

0 33 170 670 3300

3 3 3 3 3

ND b 41 ± 9 150 ± 13 479 ± 43 2 300 ±79

_ 124.2 ±27.6 88.2 ± 7.6 71.5 ± 6.4 69.7 ± 2.4

Values are the means ± standard deviation. Not detected.

Table 3. Comparison of NIV levels detected in naturally contaminated barley by competitive inhibition ELISA and by gas chromatography. NIVdetected, ppm ELISA3

GC

0.74 + 0.05 3.93 + 0.20 1.70 ±0.20 1.84 ±0.10 0.64 + 0.11 0.26 ±0.02

0.58 3.00 0.80 1.14 0.20 0.14

3 Values are the means ± the standard deviation of four replicate trials.

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36

H. Ikebuchi, R.Teshima, K. Hirai, M. Sato, M. Ichinoe andT.Terao

nivalenol by gas chromatography[6] were also subjected to CI-ELISA after extraction and sample pretreatment according to the protocols described in this paper. The results in Table 3 indicate that data obtained by CI-ELISA were in good agreement with those obtained by gas chromatography. CI-ELISA tended to give slightly higher values than gas chromatography. From the present study, it is apparent that the monoclonal antibody from clone D18.102.59 can be used effectively as an ELISA reagent for the detection of nivalenol in cereals and grains. Although the antibodies from clones J22.3.34 and L21.191.52 have similar specificity (Table 1) and association constants to Ac4-nivalenol to the antibody from clone D18.102.59, the productivity of the antibody of clone D18.102.59 was 9 and 3 times superior to those of clones J22.3.34 and L21.191.52, respectively. Therefore, we used the antibody from clone D18.102.59 for the measurement of nivalenol in barley throughout in the present study. We think this antibody is a valuable tool for the detection of nivalenol. The ELISA system in this study needs acetic anhydride treatment of the extracts to convert nivalenol to Ac4-nivalenol. Casale et al. recently succeeded in the preparing a monoclonal antibody specific for deoxynivalenol and its analogous^. They prepared immunogens by cross-linking of deoxynivalenol via the hydroxy group at the C-3 position to carrier proteins. Similar immunogens for nivalenol might help to prepare antibodies specific to nivalenol. This work was supported a research grant from the Science and Technology Agency of Japan.

References 1 2

Tanaka,T., Hasegawa, A., Matsuki,Y. & Ueno,Y. (1985) Food Add. Contamin. 2, 259-265. Lee, U.-S., Jang, H.S.,Tanaka,T.,Toyasaki, N., Sugiura, Y., Oh,Y.J., Cho, C.M. & Ueno,Y. (1986)Appl. Environ. Microbiol. 52,1258-1260.

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H. Ikebuchi*, R.Teshima andT.Terao, Division of Biochemistry and Immunochemistry, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku,Tokyo 158, Japan; M. Sato, Division of Medical Devices, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku,Tokyo 158, Japan; M. Ichinoe, Division of Microbiology, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku,Tokyo 158, Japan; K. Hirai, Department of Hygienic and Public Health, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku,Tokyo 113, Japan. * To whom correspondence should be sent.

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Production and characterization of monoclonal antibodies to nivalenol tetraacetate and their application to enzyme-linked immunoassay of nivalenol.

Three monoclonal antibodies were obtained by the fusion of mouse myeloma cells with splenocytes isolated from BALB/c mice that had been immunized with...
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