Monoclonal Antibody-Based Enzyme Linked Immunosorbent Assay of Aflatoxin B1, T-2 Toxin, and Ochratoxin A in Barley

Abstract Aflatoxin B1 (B1), T-2 toxin (T2), and ochratoxin A (OA) were assayed in a single extract from barley grain by using competitive enzyme linked immunosorbent assays (ELISAs) with monoclonal antibodies. B1 and T2 monoclonal antibodies were conjugated to horseradish peroxidase for direct competitive ELISA while an indirect competitive ELISA was used for OA determination. The competitive ELISA detected 0.1 ng/mL of B1f 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile- 0.5% KCI-6% H2S04 (89 + 10 + 1 ) extracts of barley grain either were diluted 1:10 for direct assay or were subjected to a simple liquid-liquid cleanup procedure to concentrate the extract 10:1 before assay. For cleanup, water was added to the acetonltrile extract to partition water-soluble interfering substances, and then the mycotoxins were re-extracted with chloroform. The chloroform extract was evaporated to dryness and redissolved in Tris HCI buffer for ELISA. The mean recoveries from barley spiked with 4-60 ng/g of B1( 50-5000 ng/g of T2, and 5-500 ng/g of OA were, respectively, 93.8, 80.6, and 95.8%. The mean within-assay, Inter-assay, and subsample coefficients of variation by ELISA of barley grain colonized with toxigenic fungi were <12% for Bi and OA but as high as 17% forT2.

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Mycotoxins as a risk in the grain food

Zbornik Matice srpske za prirodne nauke ◽

10.2298/zmspn0917079m ◽

2009 ◽

pp. 79-86 ◽

Cited By ~ 10

Author(s):

Jovana Matic ◽

Jasna Mastilovic ◽

Ivana Cabarkapa ◽

Anamarija Mandic

Keyword(s):

European Union ◽

Secondary Metabolites ◽

Ochratoxin A ◽

Immunosorbent Assay ◽

Toxic Effects ◽

Enzyme Linked Immunosorbent Assay ◽

The European Union ◽

The Mean ◽

Grain Foods

Mycotoxins are toxic secondary metabolites of fungi that contaminate a large variety of foods and have toxic effects on humans. The best protection against mycotoxins is to monitor their presence in food. This paper shows the screening results of mycotoxins present in 76 samples of different groups of grain foods. Samples of grain food were analyzed for contamination with aflatoxins, ochratoxin A, zearalenone, fumonisins and deoxynivalenol. Analysis were conducted using competitive enzyme-linked immunosorbent assay (ELISA). None of the samples was contaminated with aflatoxins. The most predominant mycotoxin was ochratoxin A with the mean level of 4.84 ? 4.49 ppb in 19.7% of the examined samples. Zearalenone, fumonisins, and deoxynivalenol were found in 9.21, 14.5 and 3.9% of the samples, respectively. Mycotoxin content in the investigated samples was compared with the regulations of Serbia and those of the European Union.

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Immunoassays for Analysis of Mycotoxins

Journal of Food Protection ◽

10.4315/0362-028x-47.7.562 ◽

1984 ◽

Vol 47 (7) ◽

pp. 562-569 ◽

Cited By ~ 64

Author(s):

FUN SUN CHU

Keyword(s):

Ochratoxin A ◽

Aflatoxin B1 ◽

Recent Progress ◽

Biological Fluids ◽

Aflatoxin M1 ◽

Enzyme Linked Immunosorbent Assay ◽

Specific Antibodies ◽

Protein Conjugates ◽

Advantages And Disadvantages ◽

The Past

During the past few years, several laboratories have prepared specific antibodies against aflatoxins B1, M1, B2a and Q1, ochratoxin A, T-2 toxin, and zearalenone. These antibodies were obtained from rabbits after immunizing with various mycotoxin-protein conjugates. With the availability of these antibodies, specific, simple and sensitive radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) procedures for monitoring mycotoxins and their metabolites in foods, feeds and body fluids have been developed. In this review, details are presented for the preparation of antibodies and the application of RIA and ELISA to determine aflatoxins B1 and M1, ochratoxin A and T-2 toxin in corn, peanuts, milk and other biological fluids. The sensitivity of ELISA for analysis of these mycotoxins in foods varied from 0.1 μg/L for aflatoxin M1 in milk to 5 μg/kg of aflatoxin B1 in peanuts. The advantages and disadvantages of ELISA for monitoring mycotoxins in foods and feeds are discussed. In addition, a description of recent progress on simplified clean-up procedures which may increase the sensitivity of immunoassays is presented.

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Application of ELISA to Retail Survey of Aflatoxin B1 in Peanut Butter

Journal of Food Protection ◽

10.4315/0362-028x-49.10.792 ◽

1986 ◽

Vol 49 (10) ◽

pp. 792-795 ◽

Cited By ~ 13

Author(s):

BHANU P. RAM ◽

L. PATRICK HART ◽

RICHARD J. COLE ◽

JAMES J. PESTKA

Keyword(s):

Immunosorbent Assay ◽

Aflatoxin B1 ◽

Coefficient Of Variation ◽

Simple Procedure ◽

Peanut Butter ◽

Competitive Elisa ◽

Routine Screening ◽

Enzyme Linked Immunosorbent Assay

A simple procedure was devised for the routine screening of aflatoxin B1 (AFB1) in peanut butter using enzyme-linked immunosorbent assay (ELISA). Peanut butter samples (5 g) were artificially contaminated with AFB1 and extracted by blending with 25 ml of 55% methanol and 10 ml of hexane. The extract was filtered and aqueous filtrate analyzed by a direct competitive ELISA. Recovery of AFB1 added to peanut butter samples ranged from 85 to 112%, with an average inter-well coefficient of variation of 18.4%. The inter-assay coefficient of variation was 22.7%. Using this procedure, only 3 of 63 commercial samples of peanut butter had detectable levels (>5.0 μg/kg) of AFB1.

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Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879600800111 ◽

1996 ◽

Vol 8 (1) ◽

pp. 68-75 ◽

Cited By ~ 31

Author(s):

H. E. Jensen ◽

B. Aalbaek ◽

P. Lind ◽

H. V. Krogh ◽

P. L. Frandsen

Keyword(s):

Monoclonal Antibodies ◽

Polyclonal Antibodies ◽

Fungal Species ◽

Cross Reactivity ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

Immunohistochemical Techniques ◽

Immunohistochemical Diagnosis ◽

Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

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Colonization of Barley Grain by Penicillium verrucosum and Ochratoxin A Formation in the Presence of Competing Fungi

Journal of Food Protection ◽

10.4315/0362-028x-59.12.1311 ◽

1996 ◽

Vol 59 (12) ◽

pp. 1311-1317 ◽

Cited By ~ 25

Author(s):

NANNAPANENI RAMAKRISHNA ◽

JOHN LACEY ◽

JOHN E. SMITH

Keyword(s):

Electron Microscopy ◽

Ochratoxin A ◽

Pure Culture ◽

Barley Grain ◽

Enzyme Linked Immunosorbent Assay ◽

Seed Infection ◽

Fusarium Sporotrichioides ◽

Penicillium Verrucosum ◽

Grain Surface ◽

Scanning Electron

Colonization of barley grain by Penicillium verrucosum and the formation of ochratoxin A were studied, both in pure culture and when paired with Aspergillus flavus, Fusarium sporotrichioides, and Hyphopichia burtonii, at 20° and 30°C and at 0.97, 0.95 and 0.90 aw over a 3-week period. Grain colonization was assessed on the basis of visible molding, seed infection, and numbers of CFU and by observing hyphal extension on the grain surface by scanning electron microscopy. Ochratoxin A concentrations were assayed by enzyme-linked immunosorbent assay using a monoclonal antibody. Germination of P. verrucosum spores was unaffected by the presence of other species. However, seed infection under most conditions was markedly decreased, relative to pure culture, by the presence of A. flavus and H. burtonii, but only slightly by F. sporotrichioides. The number of CFU of P. verrucosum was only slightly decreased in the presence of other species under most conditions. Generally, production of ochratoxin A by P. verrucosum was inhibited, sometimes significantly, in the presence of A. flavus and H. burtonii, but was changed only slightly by the presence of F. sporotrichioides. There was occasionally temporary enhancement in ochratoxin A production with all species during the 3-week incubation period.

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Detection of total bile acids in biological samples using an indirect competitive ELISA based on four monoclonal antibodies

Analytical Methods ◽

10.1039/c6ay03243e ◽

2017 ◽

Vol 9 (4) ◽

pp. 625-633 ◽

Cited By ~ 1

Author(s):

Shuchen Liu ◽

Yue Zhang ◽

Baoping Qu ◽

Gaofeng Qin ◽

Jinjun Cheng ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Clinical Practice ◽

Biological Samples ◽

Competitive Elisa ◽

Routine Clinical Practice ◽

Enzyme Linked Immunosorbent Assay ◽

Indirect Competitive Elisa ◽

Different Types ◽

Total Bile Acids

We investigated a newly developed indirect competitive enzyme-linked immunosorbent assay for the determination of 5 major components of TBA, which works efficiently in different types of biological samples, and may be suitable for routine clinical practice.

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Development of Indirect Competitive ELISA for Lithospermic Acid B of Salvia miltiorrhiza with Its Specific Antibodies Generated via Artificial Oil Bodies

Molecules ◽

10.3390/molecules24101952 ◽

2019 ◽

Vol 24 (10) ◽

pp. 1952 ◽

Cited By ~ 3

Author(s):

Yu-En Shih ◽

Chao-Hsiang Chen ◽

Nan-Hei Lin ◽

Jason T.C. Tzen

Keyword(s):

Metal Ion ◽

Salvia Miltiorrhiza ◽

Chinese Herb ◽

Competitive Elisa ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

Therapeutic Effects ◽

Oil Bodies ◽

Body Protein ◽

Indirect Competitive Elisa

Lithospermic acid B (LSB), the major water-soluble ingredient of Salvia miltiorrhiza (Danshen), has been shown to be an active ingredient responsible for the therapeutic effects of this traditional Chinese herb used to treat cardiac disorders. This study aimed to develop an indirect competitive enzyme linked immunosorbent assay (ELISA) for the detection of LSB. Firstly, LSB was chemically conjugated to a modified oil-body protein, lysine-enriched caleosin, recombinantly expressed in Escherichia coli. Antibodies against LSB (Ab-LSB) were successfully generated by immunizing hens with artificial oil bodies constituted with the LSB-conjugated caleosin. Western blotting showed that Ab-LSB specifically recognized LSB, but not the carrier protein, lysine-enriched caleosin. To detect LSB via indirect competitive ELISA, LSB was conjugated with bovine serum albumin (LSB-BSA) and coated on a microplate. The binding between Ab-LSB and LSB-BSA on the microplate was competed dose-dependently in the presence of free LSB with a concentration ranging from 5 to 5 × 104 ng/mL. The IC50 value was approximately determined to be 120 ng/mL for LSB regardless of its complex with a metal ion of Na+, K+ or Mg2+.

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The Development of a Quantitative ELISA for Aflatoxin B1 in Ground Peanuts Using Antibodies Isolated From the Yolk of a Laying Hen

Journal of Food Protection ◽

10.4315/0362-028x-57.11.1022 ◽

1994 ◽

Vol 57 (11) ◽

pp. 1022-1024 ◽

Cited By ~ 4

Author(s):

SUZHEN LI ◽

RONALD R. MARQUARDT ◽

ANDREW A. FROHLICH ◽

HAO XIAO ◽

JAMES R. CLARKE

Keyword(s):

Immunosorbent Assay ◽

Aflatoxin B1 ◽

Laying Hens ◽

Egg Yolk ◽

Laying Hen ◽

Competitive Elisa ◽

Enzyme Linked Immunosorbent Assay ◽

Coating Antigen ◽

Extraction Protocol

Antibodies directed against Aflatoxin B1 (AFB1) were produced in laying hens, isolated from egg yolk and applied in an enzyme-linked immunosorbent assay (ELISA) for AFB1 in ground peanuts. The ELISA sensitivity was improved by reduction of the amount of the coating antigen absorbed onto the microplate well surfaces. The main aflatoxins B1, B2, G1, G2, M1, aflatoxicol, sterigmatocystin were found to cross-react in the competitive ELISA, 100, 25, 23, 4, 0, 0, 0%, respectively. Aflatoxin B1 could be reproducibly detected and quantified in spiked ground peanuts at levels greater than 5 ppb using a hexane and methanol solvent based peanut extraction protocol.

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Transfer of aflatoxins from naturally contaminated feed to milk of Nili-Ravi buffaloes fed a mycotoxin binder

Animal Production Science ◽

10.1071/an14909 ◽

2016 ◽

Vol 56 (10) ◽

pp. 1637 ◽

Cited By ~ 5

Author(s):

N. Aslam ◽

I. Rodrigues ◽

D. M. McGill ◽

H. M. Warriach ◽

A. Cowling ◽

...

Keyword(s):

Aflatoxin B1 ◽

High Performance ◽

Mean Value ◽

Aflatoxin M1 ◽

Enzyme Linked Immunosorbent Assay ◽

Treatment Groups ◽

Low Concentrations ◽

Daily Concentration ◽

The Mean ◽

Carry Over

The objectives of this study were to observe the extent of transfer of aflatoxin B1 in feed to the aflatoxin M1 metabolite in milk in Nili-Ravi buffaloes and to evaluate the efficacy of a commercial mycotoxin binder (Mycofix, Biomin Singapore) incorporated into feed to minimise this transfer. Multiparous animals (n = 28) were randomly distributed to four groups corresponding to two treatments each with two levels of aflatoxin B1. Individual animals were exposed to naturally contaminated feed providing a total of 1475 µg/day (Groups A and B) or 2950 µg/day (Groups C and D) of aflatoxin B1. Groups B and D were given 50 g of mycotoxin binder daily mixed with feed whereas Groups A and C were kept as controls. Feed samples were analysed by reverse phase high performance liquid chromatography for aflatoxin B1 and milk samples were evaluated by enzyme-linked immunosorbent assay for the liver metabolite aflatoxin M1. The mean value of total daily aflatoxin M1 excretion for animals fed 2950 µg/day of aflatoxin B1 (112.6 µg/day) was almost double (P < 0.001) than the excretion in buffaloes fed 1475 µg/day (62.2 µg/day). The mean daily concentration of aflatoxin M1 in milk of animals from both treatment groups supplemented with 50 g/day of mycotoxin binder was 76.5 µg/day, nearly 22 µg lower than those without binder at 98.3 µg/day (s.e.d. = 5.99: P < 0.01). The interaction of binder and treatment was not significant i.e. the 50 g/day of binder was able to sequester aflatoxin B1 with the same efficiency in groups fed with high and low concentrations of aflatoxin B1. Carry over was (3.44%) lower (P = 0.001) in animals supplemented with 50 g/day of mycotoxin binder than those fed no binder (4.60%). Thus buffaloes are highly efficient at transferring aflatoxins in feed to the aflatoxin M1 metabolite in milk, whereas mycotoxin binder is capable of alleviating without preventing this contamination risk.

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