Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone

Liu, Ming-Tsiu; Ram, B. P.; Hart, L. P.; Pestka, James J.

doi:10.1128/aem.50.2.332-336.1985

Cited by 72 publications

(25 citation statements)

References 26 publications

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“…were provided. With a detection limit for ZEA of 70pg/ml in buffer, the ZEA-EIA is 5-20 times more sensitive than those reported by others (9,21,41).…”

Section: Discussionmentioning

confidence: 65%

Studies on the Application of Enzyme Immunoassays for the Fusarium Mycotoxins Deoxynivalenol, 3‐Acetyldeoxynivalenol, and Zearalenone

Usleber

Renz

Märtlbauer

et al. 1992

Journal of Veterinary Medicine, Series B

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Polyclonal antisera against zearalenone (ZEA) were produced in rabbits after immunization with ZEA-oxime coupled to human serum albumin. Using these antibodies and a ZEA-oxime-horseradish peroxidase conjugate in a competitive direct enzyme immunoassay (EIA), the detection limit for ZEA was 70 pg/ml. The relative cross-reactivities of the assay with ZEA, a-zearalenol, p-zearalenol, zearalanone, a-zearalanol, and p-zearalanol, respectively, were 100 Yo, 37.3 Yo, 7.2 %, 59.2 Yo, 5.3 %, and 3.9%, respectively. This EIA and two EIAs for deoxynivalenol (DON) and 3-acetyldeoxynivalenol(3-AcDON) (USLEHER et al., 1991) were used to analyze wheat samples. The limits of determination for DON, 3-AcDON, and ZEA in wheat were 200ppb, 50ppb, and 20ppb, respectively. The analysis of reference materials (wheat flour) containing D O N by EIA showed good agreement with the nominal values. The EIA for ZEA was in addition used to analyze biological fluids, obtained during a feeding trial. Two lactating cows were administered 25 mg and 100 mg ZEA per day, respectively, over a period of 6 days. Serum, milk, urine, and feces were assayed in the ZEA-EIA with and without sample treatment with P-glucuronidase prior to the analysis. Maximum toxin levels (ZEA-equivalents) found in milk were 0.4 and 1.2 ppb (glucuronides). The toxin concentration in milk decreased rapidly after the last toxin administration. In the urine, maximum levels of toxinglucuronide conjugates were 23 ppb and 24 ppb, respectively. The serum toxin levels corresponded to those found in milk. In the feces, mean values were 150ppb and 500ppb, respectively, no conjugated toxins were found in feces.

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“…were provided. With a detection limit for ZEA of 70pg/ml in buffer, the ZEA-EIA is 5-20 times more sensitive than those reported by others (9,21,41).…”

Section: Discussionmentioning

confidence: 65%

Studies on the Application of Enzyme Immunoassays for the Fusarium Mycotoxins Deoxynivalenol, 3‐Acetyldeoxynivalenol, and Zearalenone

Usleber

Renz

Märtlbauer

et al. 1992

Journal of Veterinary Medicine, Series B

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“…According to the molecular structure and active site of ZEN, the immunogen and coating antigen were synthesized via oxime active ester (OAE) [27], condensation mixed anhydride (CMA) [28], formaldehyde (FA) [29], and 1,4-butanediol diglycidyl ether (BDE) techniques [30].…”

Section: Synthesis Of Immunogenmentioning

confidence: 99%

Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics

Wang

Zhang

et al. 2021

International Journal of Analytical Chemistry

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Background. This study aimed to explore the zearalenone (ZEN) immunogen synthesis method, immunogenicity, and antibody characteristics and to lay a foundation for the establishment of immunoassay methods for ZEN single residue and ZEN and its analogs total residue. Methods. Based on the molecular structure and active sites of ZEN, oxime active ester (OAE), condensation mixed anhydride (CMA), formaldehyde (FA), and 1,4-butanediol diglycidyl ether method (BDE) were designed and used for immunogen (ZEN-BSA) synthesis. The immunogens were identified by infrared (IR) and ultraviolet (UV) spectra and gel electrophoresis (SDS-PAGE) and were then used to immunize Balb/c mice to prepare ZEN polyclonal antibody (ZEN pAb). The titers and sensitivity of the ZEN pAb were determined by indirect noncompetitive ELISA (inELISA) and indirect competitive ELISA (icELISA), respectively, and its specificity was assessed by the cross-reaction test (CR). Results. ZEN-BSA was successfully synthesized, and the molecular binding ratios of ZEN to BSA were 17.2 : 1 (OAE), 14.6 : 1 (CMA), 9.7 : 1 (FA), and 8.3 : 1 (BDE), respectively. The highest inELISA titers of ZEN pAb of each group were 1 : (6.4 × 103) (OAE), 1 : (3.2 × 103) (CMA), 1 : (1.6 × 103) (FA), and 1 : (1.6 × 103) (BDE), respectively. The 50% inhibition concentrations (IC50) for ZEN by icELISA of each group were 11.67 μg/L (OAE), 16.29 μg/L (CMA), 20.92 μg/L (FA) and 24.36 μg/L (BDE), respectively. ZEN pAb from the mice immunized with ZEN-BSA (OAE) and ZEN-BSA (CMA) had class broad specificity to ZEN and its analogs. The CRs of ZEN pAb with α-ZAL, β-ZAL, α-ZOL, β-ZOL, and ZON were 36.53%, 16.98%, 64.33%, 20.16%, and 10.66%, respectively. ZEN pAb from the mice immunized with ZEN-BSA (FA) and ZEN-BSA (BDE) had high specificity for ZEN. The CRs of ZEN pAb with its analogs were all less than 1.0%. Conclusion. This study demonstrated that the preparation of the class broad-specificity antibodies of ZEN and its analogs can be achieved by immunizing animals with the immunogen ZEN-BSA prepared by the OAE method, while the preparation of highly specific antibodies can be achieved by immunizing animals with the immunogen ZEN-BSA prepared by the FA method. These findings lay the material and technical foundation for immunoassay of ZEN single residue and ZEN and its analogs total residue.

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“…CI-ELISA for spiked corn extracts. The procedures for extraction from corn and spiking were similar to that described by Liu et al (15). Briefly, zearalenonefree homogeneous ground corn was extracted with 5 volumes (wt/vol) of methanol-water (70:30) for 5 min, with constant stirring.…”

Section: Methodsmentioning

confidence: 99%

Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone

Yuan

Clarke

Zhou

et al. 1997

Appl Environ Microbiol

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The heavy-chain and kappa light-chain variable region genes of an antizearalenone hybridoma cell line (2G3-6E3-2E2) were isolated by PCR and joined by a DNA linker encoding peptide (Gly 4 Ser) 3 as a single-chain Fv (scFv) DNA fragment. The scFv DNA fragment was cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with E tag and phage M13 p3 in Escherichia coli TG1. In the presence of helper phage M13K07, the scFv fusion protein was displayed on the surfaces of recombinant phages. High-affinity scFv phages were enriched through affinity selection in microtiter wells coated with zearalenone-ovalbumin conjugate. The selected recombinant phages were used to infect E. coli HB2151 for the production of soluble scFv antibodies. One selected clone (pQY1.5) in HB2151 secreted a soluble scFv antibody (QY1.5) with a high zearalenone-binding affinity (concentration required for 50% inhibition of binding, 14 ng/ml), similar to that of parent monoclonal antibody in a competitive indirect enzyme-linked immunosorbent assay. However, scFv QY1.5 exhibited higher cross-reactivity with zearalenone analogs and had greater sensitivity to methanol destabilization than the parent monoclonal antibody did. Nucleotide sequence analyses revealed that the light-chain portion of scFv QY1.5 had a nucleotide sequence identity of 97% to a mouse germ line gene V K 23.32 in mouse kappa light-chain variable region subgroup V, whereas the heavy-chain nucleotide sequence was classified as mouse heavy-chain subgroup III (D) but without any closely related members having highly homologous complementarity-determining region sequences. The potential of soluble scFv QY1.5 for routine screening of zearalenone and its analogs was demonstrated with zearalenone-spiked corn extracts. Zearalenone [6-(10-hydroxy-6-oxo-trans-1-undecenyl)-␤resorcylic acid lactone] is a mycotoxin produced by members of the genus Fusarium after infection of corn and small grains (8, 19, 21). When fed to animals, the compound causes hyperestrogenism with symptoms such as enlargement of the uterus and nipples, vulvar swelling, vaginal prolapse, and infertility (10, 20). As a consequence of these estrogenic effects, there is a need for routine screening of agricultural commodities used for human and animal consumption. Methods for the analysis of zearalenone in food and feeds include thin-layer chromatography (5, 30, 31), gas-liquid chromatography (30, 34), high-pressure liquid chromatography (11, 30, 36), and, more recently, immunoassays (2, 4, 15, 32, 35, 37). Compared with other methods, immunoassays have several advantages for rapid field tests, including high specificity, sensitivity, facile sample preparation, and ease of use (27). However, the development of antibodies requires the use of animals, specialized cell culturing facilities, and an extensive commitment of time and labor. Production of monoclonal antibodies from hybridoma cell lines also requires specialized cell culture facilities and usually is time-consuming. Advances in the field of recombinant ant...

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Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone

Cited by 72 publications

References 26 publications

Studies on the Application of Enzyme Immunoassays for the Fusarium Mycotoxins Deoxynivalenol, 3‐Acetyldeoxynivalenol, and Zearalenone

Studies on the Application of Enzyme Immunoassays for the Fusarium Mycotoxins Deoxynivalenol, 3‐Acetyldeoxynivalenol, and Zearalenone

Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics

Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone

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