Preparation and Characterization of Anti-Fumonisin Monoclonal Antibodies

Fukuda, Satoshi; Nagahara, Ayumu; Kikuchi, Mamoru; Kumagai, Shinya

doi:10.1271/bbb.58.765

Cited by 21 publications

(27 citation statements)

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“…Although several mAbs for fumonisins have been made in the past decade, most of the mAb-based ELISAs for fumonisins were not very sensitive a Two samples were spiked at each toxin level, and each sample was run in triplicate. (Azcona-Olivera et al, 1992;Fukuda et al, 1994). The mAb-based ELISA established in the present study is considerably more sensitive than others.…”

Section: Discussionsupporting

confidence: 46%

“…Immunoassays have been widely used in recent years for the detection of mycotoxins, including fumonisins (Azcona-Olivera et al, 1992a,b;Fukuda et al, 1994;Yeung et al, 1996;Yu & Chu 1996, 1998. Although several mAbs for fumonisins have been made in the past decade, most of the mAb-based ELISAs for fumonisins were not very sensitive a Two samples were spiked at each toxin level, and each sample was run in triplicate.…”

Section: Discussionmentioning

confidence: 99%

“…Immunochemical methods have been used widely for mycotoxin analysis during the past few years. Both monoclonal (mAb) and polyclonal antibodies specific to FmB1 have been generated recently (Azcona-Olivera et al, 1992a,b;Fukuda et al, 1994;Usleber et al, 1994;Yeung et al, 1996;Yu & Chu, 1996, 1998. The availability of specific and sensitive mAb provides an unlimited supply of immunochemical reagents for enzyme-linked immunosorbent assay (ELISA) and allows further investigation of antibody structure.…”

Section: Introductionmentioning

confidence: 99%

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Production and Characterization of Monoclonal Antibodies against Fumonisin B1

Yu¹,

Chu²

1999

Food and Agricultural Immunology

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Monoclonal antibodies (mAb) against fumonisin B1 (FmB1) were produced from a stable hybridoma cell line, 9C11E6, generated by the fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with FmB1-keyhole limpet hemocyanin. The 9C11E6 mAb belong to the immunoglobulin G1 (kappa chain) isotype with the highest specificity for FmB3. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an indirect competitive ELISA (idc-ELISA) were established for antibody characterization and toxin analysis. The concentrations causing 50% inhibition (IC 50) of binding of FmB1horseradish peroxidase with the antibodies by FmB1, FmB2, and FmB3 in the dc-ELISA were found to be 29.1, 17.5, and 4.1 ng ml-1 , respectively. The IC 50 values of the binding of 9C11E6 mAb to the solid-phase FmB1-ovalbumin by free FmB1, FmB2, and FmB3 in the idc-ELISA were found to be 20.1, 13.2, and 16.8 ng ml-1 , respectively. The average recovery of FmB1 (50-2000 ng g-1) added to cornmeal and subsequently extracted with phosphatebuffered saline in the dc-ELISA was found to be 71.3%. The mAb did not react with the hydrolyzed FmB1, sphingosine, sphinganine, or tricarballylic acid, but showed weak crossreactivity (about 6.8% of FmB1) with the Alternaria alternata f. sp. lycopersici toxin TA.

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Section: Discussionsupporting

confidence: 46%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Production and Characterization of Monoclonal Antibodies against Fumonisin B1

Yu¹,

Chu²

1999

Food and Agricultural Immunology

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“…With the advance of biotechnology, antibodies have been produced against the fumonisins and have been used in both enzyme-linked immunoassays (ELISAs) and immunoaffinity columns. Immunochemical methods can produce results more quickly than the traditional methods (17)(18)(19)(20)(21)(22). Immunochemical methods are often the methods of choice for both mycotoxin monitoring and surveillance studies, which require rapid analysis of a large number of samples.…”

Section: Immunochemical Methods For Fumonisins In Corn 327mentioning

confidence: 99%

Immunochemical Methods for Fumonisins in Corn

Trucksess

1996

ACS Symposium Series

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An enzyme-linked immunosorbent assay (ELISA) and an immunoaffinity column (IAC) cleanup procedure have been successfully applied to the determination of fumonisins in corn. The performance of the ELISA was evaluated by comparison to a reference high-performance liquid chromatographic (HPLC) method. The IAC procedure was coupled with HPLC determination. The recoveries of fumonisin B 1 from corn spiked at the 1,2, and 4μg/g levels were 73-106, 79-83, and 64-92% for the ELISA, IAC, and HPLC methods, respectively. The accuracy and precision of the methods compared favorably. In the comparative studies using naturally contaminated corn samples, the ELISA results were 2-100% higher than those determined by HPLC. The immunoaffinity procedure results were about 71% of the levels observed using HPLC.The fumonisins are a group of structurally related mycotoxins, which are secondary metabolites produced on corn by Fusarium moniliforme (1,2), Fusarium proliferatum (3), and several other flingi (4,5). Of the known naturally occurring fumonisins, fumonisins B l (FB X ) and Bj (F^ ) are the most abundant (6). In the United States the FB 1 fFB 2 ratio in corn is about 3:1 (7). was found to cause equine leukoencephalomalacia (2), porcine pulmonary edema (8), and rodent hepatotoxicity (9). Cattle and poultry can also be affected, but are not as susceptible to the mycotoxin as horses and swine. The Mycotoxin Committee of the American Association of Veterinary Laboratory Diagnosticians recommends that the total intake of FB! be limited to less than 5 μg/g in the non-roughage diet of horses, 10 μg/g in the total diet of swine and 50 μg/g in the feed for cattle and poultry (10).

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“…An alternative approach has been the use of enzyme-linked immunosorbent assays (ELISAs), which generally have required no clean-up other than sample filtration and dilution. Several monoclonal antibody (MAb) and polyclonal antibody (PAb)-based ELISAs are available for detection of the intact and hydrolyzed fumonisins (Azcona-Olivera et al, 1992;Fukuda et al, 1994;Pestka et al, 1994;Shelby et al, 1994;Usleber et al, 1994;Chu et al, 1995;Elissalde et al, 1995;Maragos et al, 1996b;Yu & Chu, 1996). The most sensitive assays, with limits of detection in the low ppb range, have relied upon PAbs rather than MAbs (Usleber et al, 1994;Yu & Chu, 1996).…”

Section: Introductionmentioning

confidence: 99%

Detection of the mycotoxin fumonisin B₁by a combination of immunofluorescence and capillary electrophoresis

Maragos

1997

Food and Agricultural Immunology

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The specificity of antibodies has been combined with the speed and resolving power of capillary electrophoresis for application to the analysis of the mycotoxin fumonisin B 1 (FB 1). The assay was based upon the competition between unlabeled FB 1 (i.e. from a sample) and a fluorescein-labeled FB 1 reagent (FB 1-FL). The FB 1-FL was prepared by derivatizing FB 1 with fluorescein isothiocyanate and was purified with affinity columns consisting of a monoclonal antibody (MAb) directed against fumonisins (clone P2A5-3-F3) coupled to agarose. The purified FB 1-FL was subjected to capillary zone electrophoresis. Addition of purified MAb to FB 1-FL before separation resulted in the formation of a complex (MAb.FB 1-FL) with resulting quenching of fluorescence and decrease in the intensity of the FB 1-FL peak. When unlabeled FB 1 was also added to the reaction mixture the FB 1 and FB 1-FL competed for the limited amount of antibody present causing the FB 1-FL peak to increase in direct proportion to the amount of unlabeled FB 1 present. Fumonisin standards could be analyzed with this technique with a total analysis time of 6 min, 2 min of which was required for washing the capillary between analyses. The concentration of unlabeled FB 1 required to obtain 50% of the maximum fluorescence (IC 50) was highly dependent upon the antibody concentration and ranged from 58 to 4170 ng ml-1 at 15-75 µg m-1 of antibody. The optimum performance was seen with 25-50 µg ml-1 of antibody, with IC 50 's between 500 and 1700 ng ml-1 of FB 1. The technique was applied to a limited number of corn samples spiked with high levels of FB 1 (> 10 ppm). This technology holds considerable promise for the rapid analysis of mycotoxins in foods.

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Preparation and Characterization of Anti-Fumonisin Monoclonal Antibodies

Cited by 21 publications

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Production and Characterization of Monoclonal Antibodies against Fumonisin B1

Production and Characterization of Monoclonal Antibodies against Fumonisin B1

Immunochemical Methods for Fumonisins in Corn

Detection of the mycotoxin fumonisin B₁by a combination of immunofluorescence and capillary electrophoresis

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Preparation and Characterization of Anti-Fumonisin Monoclonal Antibodies

Cited by 21 publications

References 0 publications

Production and Characterization of Monoclonal Antibodies against Fumonisin B1

Production and Characterization of Monoclonal Antibodies against Fumonisin B1

Immunochemical Methods for Fumonisins in Corn

Detection of the mycotoxin fumonisin B1by a combination of immunofluorescence and capillary electrophoresis

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Product

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Detection of the mycotoxin fumonisin B₁by a combination of immunofluorescence and capillary electrophoresis