Letters in Applied Microbiology 1989,9, 133-135 MOM/022

A monoclonal antibody-based enzyme immunoassay for the detection of T-2 toxin at picogram levels

R. HACK, E. MARTLBAUER & G. TERPLAN Znstitute for Hygiene and Technology of Food of Animal Origin, Veterinary Faculty, University of Munich, 8000 Munich 22, Federal Republic of Germany

Received 13 June 1989 and accepted 4 July 1989

HACK, R., MARTLBAUER, E. & TERPLAN, G . 1989. A monoclonal antibody- based enzyme immunoassay for the detection of T-2 toxin at picogram levels. Let- ters in Applied Microbiology, 9, 133-135.

Thirteen monoclonal antibodies reactive with HT-2 were prepared by using a HT-2 hemisuccinate coupled to human serum albumin as antigen for the immunization of BALB/c mice. In a competitive enzyme immunoassay on a double antibody solid phase using HT-2 hemisuccinate coupled to horseradish peroxidase as enzyme linked toxin all antibodies reacted much better with T-2 toxin and acetyl T-2 than with HT-2. Eleven antibodies showed almost the same sensitivity and specificity, and one of these, designated 3E2, is extensively described. Its cross-reactivities with HT-2, T-2 toxin, acetyl T-2, is0 T-2, T-2 tetraol tetraacetate and T-2 trio1 were 1.0, 140.2, 161.2, 0.32, 0.14 and 0.016, respectively. Two other antibodies, designated 2A4 and 2A5, behaved quite differently. The cross-reactivities of antibody 2A4 with these toxins were: 1.0, 113.9, 374.4, 1.35, 0.34 and 0.023, respectively; for antibody 2A5 they were 1.446.1, 155.4, 8.31,0.9 and 0.08, respectively. All antibodies proved to be IgG1. By using the antibody 3E2 a highly sensitive and very specific enzyme immunoassay for the detection of T-2 toxin was developed. The detection limit for T-2 toxin was 5 pgJml(O.25 pdassay).

The mycotoxin T-2 toxin, a member of the tri- chothecene group, has been associated with out- breaks of severe mycotoxicoses in the past and is known as a contaminant of foods and feeds (Snyder 1986). Therefore, besides the widely used physicochemical procedures, immunologi- cal methods including monoclonal antibody- based enzyme immunoassays have been developed for its rapid and specific detection (Gendloff e t al. 1987; Hack e t al. 1987; Good- brand et al. 1987; Chiba e t al. 1988).

For the immunization of mice, antigens pro- duced by conjugation of T-2 toxin to a protein carrier through the functional hydroxyl group at the C-3 position of the molecule have gener- ally been used. The resulting monoclonal anti-

Address for correspondence: Dr R. Hack, Institut fur Hygiene und Technologie der Lebensmittel tieris- chen Ursprungs, Universitat Miinchen, Schellingstr. lO/III, D-8000 Munchen 40, Federal Republic of Germany.

bodies, however, have not been as sensitive as the corresponding polyclonal antisera in enzyme immunoassays for T-2 toxin.

In this study an alternative approach was made by coupling HT-2 dihemisuccinate to human serum albumin. The very high reactivity of the resulting monoclonal anti-HT-2 antibody 3E2 with T-2 toxin was utilized in an enzyme immunoassay for the detection of T-2 toxin at picogram levels.

Materials and Methods

PREPARATION OF HT-2 HEMISUCCINATE PROTEIN CONJUGATES

The hemisuccinate of HT-2 was prepared according to Chu et al. (1979) and purified by thin layer chromatography on Silica Gel G-60 (E. Merck, Darmstadt, Federal Republic of Germany) with toluol-ethylacetate (25 : 75) in

134 R. Hack et al. an unequilibrated tank. Mass spectrum (MS) of the HT-2 derivative was carried out on a quad- rupole mass spectrometer (model 1020, Finni- gan MAT, Bremen) by methane chemical ionization direct-exposure probe analysis according to Martlbauer et al. (1988).

The antigen, HT-2 hemisuccinate coupled to human serum albumin, was prepared following the method used for the production of HT-2- horseradish peroxidase conjugate (HT-2-HRP) as described elsewhere (Hack et al. 1987).

PRODUCTION A N D CHARACTERIZATION OF MONOCLONAL ANTIBODIES

Female BALB/c mice were immunized by repeated intraperitoneal injections of antigen. The splenocytes were fused and the antibody- secreting hybridomas were detected and propa- gated as described (Hack et al. 1987, 1989). The cross-reactivity studies, as well as the determi- nation of the immunoglobulin class and sub- class, were performed according to previous papers (Hack et al. 1987, 1989). Ascites was purified by double precipitation with saturated ammonium sulphate solution followed by column chromatography with Affi-Gel Protein A MAPS I1 Kit according to the instructions of the producer (Biorad, Munchen, Federal Republic of Germany).

ENZYME IMMUNOASSAY FOR THE

DETECTION OF T - 2 TOXIN

Immunoplates were incubated as usual. After washing, each well was incubated with loop1 of an antibody solution containing 15 ng antibody 3E2/ml for 1 h. Following another washing pro- cedure HT-2-HRP (50 pl, dilution 1 : 20000 in phosphate-buffered saline containing 1% FCS) and T-2 toxin standards (50 p1 containing 10% methanol) were incubated simultaneously for 1 h. Then the assay proceeded as usual. A typical standard curve for the determination of T-2 toxin is shown in Fig. 1.

Results and Discussion

To conjugate HT-2 to a carrier a reactive car- boxyl moiety had to be introduced on to the molecule by reaction with succinic anhydride. Only one derivative was detected by thin layer chromatography. The R, values for HT-2 and its derivative, HT-2 hemisuccinate, under the

“ I \

0 \

\ \

0 I I 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 I I I l l l 111 I 1 1 1

0.1 I 10 100 1000 T-2 toxin concentration (pglrnl)

Fig. 1. Standard curve for the detection of T-2 toxin with the monoclonal anti-HT-2 antibody 3E2.

conditions described above were 0.33 and 0.65, respectively.

Since HT-2 has two hydroxy groups which can be converted to the corresponding esters the derivative was further characterized by mass spectral analysis. The chemical ionization mass spectrum revealed m/z 625 as the protonated molecular ion (the molecular weight of HT-2 dihemisuccinate is 624) and the adduct ions m/z 653 (M + C,H,)+ and m/z 665 (M + C,H5)+ which confirm the formation of HT-2 dihemi- succinate. Thirteen positive hybridomas were found in the first test for anti-HT-2 antibody production. Further characterization revealed that 11 antibodies behaved so similarly in terms of specificity and affinity that only one of them, designated 3E2, is herein described. Two others, 2E4 and 2E5, however, showed differences espe- cially in their affinity to HT-2, but also in their specificity relative to HT-2 (Table 1).

All secreted antibodies proved to be IgG1. The cross-reactivity studies showed that the three antibodies herein presented (Table 1) reacted much better with T-2 toxin and acetyl T-2 than with HT-2 and other trichothecenes. This finding was not unexpected since HT-2 dihemisuccinate used for the preparation of the antigen chemically resembles more T-2 toxin and especially acetyl T-2 than HT-2. The absol- ute necessity of an acylic substituent at position C-8, either an isovaleroxy or an acetoxy group, was confirmed by the absence of significant

Enzyme immunoassay for T-2 toxin Table 1. Specificity of the monoclonal antibodies 3E2, 2A4 and 2A5 relative to HT-2

135

Cross-reactivity relative to HT-2

Toxin 3E2 2A4 2A5

HT-2 1.0 (0.07) 1 .o 1 .o T-2 toxin 140.2 (1.0) 113.9 45.1 Acetyl T-2 161.1 (1.14) 374.4 155.4 ISO T-2 0.32 (0.02) 1.35 8.31 T-2 tetraol tetraacetate 0.14 (0.001) 0.34 0.9 T-2 trio1 0.016 (0.OOOl) 0.023 0.08 50% inhibition concn. of HT-2 6.36 17.68 253.2

(nmolP) Numbers in parentheses in the first row indicate the cross-reactivity relative to

T-2 toxin. Other trichothecenes tested (neosolaniol, T-2 tetraol, 1 5-acetoxyscirpenol, scir-

pentriol, diacetylverrucarol, verrucarol, fusarenon X, 3a-acetyldeoxynivalenol, deoxynivalenol) were not reactive with the antibodies.

cross-reactivities with trichothecenes missing these groups.

Since the aim of this study was the install- ation of an enzyme immunoassay for the detec- tion of T-2 toxin we utilized the high reactivity of the most sensitive antibody 3E2 with T-2 toxin. A concentration of 15ng antibody 3E2 per ml was sufficient to cause a Ads0 reading of about 1.0 after 10 min reaction time of sub- strates. The detection limit for T-2 toxin, deter- mined as toxin concentration at 80% of absolute A,,,, was at 5 p d m l (0.25 pg/assay). The linear part of the standard curve was in the range of 5-100 pg/ml, the 50% inhibition con- centration was at 20 pg/ml, and the intra-assay coefficients of variation (n = 4) for various stan- dard concentrations were usually below 5%.

Since only acetyl T-2 showed significant cross-reactivity relative to T-2 toxin the assay can be considered to be very specific for T-2 toxin. It seems reasonable to assume that the detection limit i$ a t least 10 times higher than the most sensitive antibody recently reported (Chiba et al. 1988).

The authors acknowledge M. Gareis, Institute for Medical Microbiology, Infectious and Epi- demic Diseases, Veterinary Faculty, University of Munich for the mass spectrum and thank B. Bloching for excellent technical assistance.

References CHIBA, J., KAWAMURA, O., KAJII, H., OHTANI, K.,

NAGAYAMA, S. & UENO, Y. 1988 A sensitive enzyme-linked immunosorbent assay for detection of T-2 toxin with monoclonal antibodies. Food Additives and Contaminants 5,629-639.

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GOODBRAND, LA., STIMSON, W.H. & SMITH, J.E. 1987 A monoclonal antibody to T-2 toxin. Letters in Applied Microbiology 5,97-99.

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HACK, R., KLAFFER, U. & TERPLAN, G. 1989 A mono- clonal antibody to the trichothecene mycotoxin dia- cetoxyscirpenol. Letters in Applied Microbiology 8,

M ~ T L B A U E R , E., CAREIS, M. & TFRPLAN, G. 1988 Enzyme immunoassay for the macrocyclic tri- chothecene roridin A: production, properties, and use of rabbit antibodies. Applied and Environmental Microbiology 54,225-230.

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