[Show abstract][Hide abstract] ABSTRACT: Eighty four samples of commercial infant foods in Libya were examined for microbiological quality. Bacillus cereus, B. stearothermophilus, B. licheniformis, Staphylococcus xylosus, S.lentus, Enterobacter sakazakii, E. aerogenes were isolated from the samples. Over 64.3 % of the samples contained high counts of Bacillus spp (2 log 10 CFU/g), 42.9% Staphylococcus spp (2 log 10 CFU/g) and 26.3% Enterobacteriaceae (2 log 10 CFU/g). The moulds isolated were mainly of the genera, Aspergillus and Penicillium. In relation to antibiotic resistance Bacillus spp showed the highest level of resistance to bacittracin (63.6%), ampicillin (54.5%), cephalosporin (36.4%), penicillin (18.1%) and nalidixine acid (18.2%). Corresponding values for Staphylococcus spp were bacitracin 60%, erythromycin 30%, penicillin 30%, cepha-losporin 10%, nalidixic acid 10% and ampicillin 10%, respectively. Enterobacteriaceae strains were resistant to bacitracin (100%), erythromycin (62.5%), ampicillin (37.5%), cephalosporin (25%) and nalidixine acid (12.5 %). Bacillus spp, Staphylococcus spp and Enterobacteriaceae were susceptible to chloramphenicol, kanamycin, gentamicin and streptomy-cin.
[Show abstract][Hide abstract] ABSTRACT: Eight polyherbal products sold in Malaysia were tested for mycoflora and the extracts analyzed for aflatoxins and ochratoxin A. Fungal count was low (less than 400 cfu/g) in all samples. Aspergillus. spp. were isolated from all samples, but none of the isolates was mycotoxigenic. Other fungi isolated were Eurotium. spp., Cladosporium. spp., Scopulariopsis. spp., Phialophora. spp., Fonseceae. spp., Penicillium. spp. and Paecilomyces. spp. Only one product was contaminated with mycotoxin: ochratoxin A. The herbal extracts were also tested for cytotoxicity on human cell lines Hep2 and HFL1 using the MTT assay. All extracts were cytotoxic to both cell lines at a concentration of 500 µg/ml. Four extracts were cytotoxic to both cells at 50 µg/ml but showed varying effects at 5 µg/ml. Five products, including those that were cytotoxic, interacted positively with DNA using the DNA–methylgreen assay. In vitro. cytotoxicity tests showed that half of the products were cytotoxic and interacted with DNA.
[Show abstract][Hide abstract] ABSTRACT: Polyclonal antibody sensitive to Listeria was assayed for the detection of Listeria using two different methods, direct and sandwich enzyme linked immunosorbent assays (ELISAs). The direct ELISA uses anti-goat IgG antibody conjugated with horse-radish peroxidase, while the sandwich ELISA uses two antibodies both specific to Listeria antigens, one coated onto the microtitre plate and the other conjugated to horse-radish peroxidase. The results obtained show that the direct ELISA is superior to the sandwich ELISA in two distinct ways: (i) with direct ELISA the non-Listeria gave readings <0.2, whereas with sandwich ELISA it gave readings of 0.3–0.4; (ii) the direct ELISA is more cost-effective than the sandwich ELISA.
[Show abstract][Hide abstract] ABSTRACT: Aflatoxins B1, B2, G1 and G2 can be easily and rapidly detected in aqueous solutions using an affinity chromatography column coupled to a monoclonal antibody specific for the toxin molecules. Water: methanol extracts of food uncontaminated with aflatoxins were spiked with aflatoxins, diluted with water, and passed through the affinity matrix. The monoclonal antibody is bound to the aflatoxin, which can then be released by using a small volume of methanol. This results in both concentration and separation of the aflatoxin present in solution. As little as 5 ng of aflatoxin can be visualized in the methanol eluate if passed over a small florisil tip under ultraviolet light, while 0.5 ng can be detected in the methanol eluate if analysed by high performance liquid chromatography (HPLC). Thus, this system can be used to test for aflatoxins in contaminated samples by spot testing (> 5 ng) or as a means of HPLC clean-up for quantitative analysis at subnanogram levels. The advantages of this immunological assay in relation to other immunoassays and traditional methods are discussed.
International Journal of Food Science & Technology 06/2007; 23(5):479 - 485. · 1.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Forty-nine samples of raw cow's milk and 20 samples of fresh white soft cheese were collected directly from 20 local dairy factories in the north-west of Libya and analysed for the presence of aflatoxin M1 (AFM1). The samples were analysed using a high-performance liquid chromatography technique for toxin detection and quantification. Thirty-five of the 49 milk samples (71.4%) showed AFM1 levels between 0.03 and 3.13 ng ml(-1) milk. Multiple analyses of five milk samples free of AFM1 artificially contaminated with concentrations of AFM1 at 0.01, 0.05, 0.1, 1.0 and 3.0 ng ml(-1) showed average recoveries of 66.85, 72.41, 83.29, 97.94 and 98.25%, with coefficients of variations of 3.77, 4.11, 1.57, 1.29 and 0.54%, respectively. Fifteen of 20 white soft cheese samples (75.0%) showed the presence of AFM1 in concentrations between 0. 11 and 0.52 ng g(-1) of cheese. Multiple assays of five cheese samples free of AFM1 spiked with different concentration of AFM1 (0.1, 0.5, 1.0 and 3.0 ng g(-1)) showed average recoveries of 63.23, 78.14,83.29 and 88.68%, with coefficients of variation of 1.53, 9.90, 4.87 and 3.79%, respectively. The concentrations of AFM1 were lower in the cheese products than in the raw milk samples.
Food Additives and Contaminants 07/2004; 21(6):592-7. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Twenty-five varieties of dates (Phoenix dactylifera) were examined at different maturation stages for total microbial counts, aflatoxins and aflatoxigenic Aspergillus sp. and lactic acid bacteria. The samples were examined as fresh and under simulated storage condition of high humidity. Microbial counts were high at the first stage of maturation (Kimri) and increased sharply at the second stage (Rutab), then decrease significantly at the final dried stage of maturation (Tamr). Aflatoxins were detected in 12% of the samples although aflatoxigenic Aspergillus were detected in 40% of the varieties examined, all at Kimri stage only. Lactic acid bacteria were present only at the Rutab stage in some varieties including all varieties in which aflatoxins or aflatoxigenic Aspergillus were detected. No aflatoxins or aflatoxigenic Aspergillus were detected at the final edible stage of maturation.
International Journal of Food Microbiology 12/2002; 79(1-2):113-9. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A technique for the detection of aflatoxins in pistachio and cashew nuts using immunoaffinity column clean-up with HPLC and fluorescent detection is presented. Recoveries were in the range of 79–99% for pistachio samples artificially contaminated with 10g total aflatoxins kg–1 of food sample. For cashew samples recoveries ranged from 80–106%. This method is proposed as an accurate technique for aflatoxin detection in the range of g aflatoxins kg–1 nuts.
[Show abstract][Hide abstract] ABSTRACT: Five separate monoclonal antibodies were produced against whole cell extracts ofAspergillus flavus and ELISA procedures used to characterise the reactivity of the antibodies to various fungal extracts. All five antibodies
were specific to the aflatoxin producing fungi,A, flavus andA. parasiticus, and indicated no cross reactivity with otherAspergillus species, genera of several fungi or with other components which may be found in food samples whereA. flavus may be found.
[Show abstract][Hide abstract] ABSTRACT: The AflatestR immunoaffinity method has been successfully used to measure total aflatoxins in experimental situations. A high level of correlation was achieved between the immunoaffinity separation and fluorimetric quantification of total aflatoxins and traditional solvent extraction and quantification by HPLC methods.
[Show abstract][Hide abstract] ABSTRACT: Immunoassays provide rapid, specific, sensitive and inexpensive methods for analysing mycotoxins but have generally been tested individually. Mycotoxigenic fungi rarely occur in pure culture in nature, and different mycotoxins may occur together. It would therefore be advantageous if several immunoassays for different mycotoxins could utilize a single extract. Monoclonal antibodies specific for ochratoxin A, aflatoxin B1 and T-2 toxin, raised at the University of Strathclyde, allow detection limits of 1 ng/ml ochratoxin A, 0.1 ng/ml aflatoxin B1 and 10 ng/ml T-2 toxin, when used in competitive enzyme-linked immunosorbent assays. These antibodies have been used to assay ochratoxin A. aflatoxin B1 and T-2 toxin in a single acetonitrile: 0.5% KCl:6% H2SO4 (89:10:1) extract of cereal grain. Extracts were either diluted 1:10 for direct assay or subjected, before assay, to a simple liquid-liquid clean-up procedure, which removed interfering substances and resulted in a 5:1 concentration. Recoveries from barley to which mycotoxins had been added averaged 95.8% for ochratoxin A, 93.8% for aflatoxin B1 and 80.6% for T-2 toxin, and the detection limits were 5 ng/g for ochratoxin A, 4 ng/g for aflatoxin B1 and 50 ng/g for T-2 toxin. Mean coefficients of variation within and between assays and between subsamples were less than 12% for ochratoxin A and aflatoxin B1 but up to 17% for T-2 toxin in assays of barley inoculated with toxigenic fungi.(ABSTRACT TRUNCATED AT 250 WORDS)
[Show abstract][Hide abstract] ABSTRACT: A monoclonal antibody (mAb) has been produced to aflatoxin B1 (AF B1) after successful immunization of mice and fusion of sensitized spleen cells with myeloma cancer cells. The mice were immunized with AF B1-oxime-protein conjugate. Positive mAbs were screened using an indirect ELISA specific for AF B1. The selected mAb was then developed in direct competitive ELISA and immunoaffinity column chromatography methods for aflatoxin detection in foods and feeds. Both assays are rapid, sensitive, specific and require only the minimum of sample preparation. Both immunological assays have now been commercialized and are produced in convenient ready-made kit formats.
Letters in Applied Microbiology 05/1990; 10(4):167-9. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Specific monoclonal antibodies (MABs) against aflatoxins, ochratoxin A, zearalenone, diacetoxyscirpenol and T-2 toxin have been prepared in various laboratories by the application of hybridoma technology to mycotoxins. These antibodies can be selected for sensitivity, reduced cross-reactivity, reliability and ease of production. When a suitable antibody is chosen it can then be used in a rapid immunological method such as an enzyme-linked or radio-immunoassay or immunoaffinity chromatography system. These assays have a lower limit of mycotoxin detection in the ng/ml range and have been applied to the determination of mycotoxins in samples such as maize, peanuts, peanut butter, milk and porcine kidneys. Using these immunoassay techniques, sample preparation has generally been simplified to a matter of solvent extraction of mycotoxins from the sample followed by dilution; under these conditions, levels of 1-5ug of mycotoxins/kg of sample can be found. The application and advantages of MABs to mycotoxins and the use of these antibodies in various assay techniques is discussed.
[Show abstract][Hide abstract] ABSTRACT: A simple and rapid indirect enzyme-linked immunosorbent assay was developed for the quantitative determination of ochratoxin A in barley after the successful production of a high affinity, specific monoclonal antibody. A rapid sample cleanup was achieved by extracting ochratoxin A from barley with chloroform and partitioning the toxin into bicarbonate buffer; the buffer solution was then added directly to the assay plate and ochratoxin A content was assessed. Recoveries were greater than 85% and detection limits were 5 micrograms ochratoxin A/kg barley.
Journal - Association of Official Analytical Chemists 01/1988; 71(5):961-4.
[Show abstract][Hide abstract] ABSTRACT: By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.
Applied and Environmental Microbiology 04/1987; 53(3):514-8. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A simple procedure was validated for the routine immunochemical analysis of AFB1 in peanut kernels, peanut butter and maize. The specificity, affinity and sensitivity of the monoclonal antibody (McAb) employed was such that minimal sample preparation was required. The enzyme immunoassay (EIA) had a sensitivity for standard AFB1 of 0·2 ng ml−1 with a working range up to 30 ng ml−1 and was not significantly affected by matrix interference of samples. Essential protocol features were (1) blending substrate with methanol: water; (2) filtering blended sample; and (3) analysis of filtrate by EIA after dilution with buffer. Average recoveries of AFB1 spiked samples at levels of 6–400 ppb were 90–112·5%. Using laboratory prepared samples contaminated with Aspergillus flavus there was high positive correlation (r = 0·97) when EIA results were compared with thin layer chromatography (TLC) techniques.
[Show abstract][Hide abstract] ABSTRACT: A monoclonal antibody (McAb) specific for ochratoxin A (OA) has been developed. The McAb has low cross-reactivity for ochratoxin α, although cross-reactivity with the synthetic ochratoxin C was high. The affinity dissociation constant of the McAb was found to be 6-2 × 107 M-1 by ELISA. A competitive enzyme immunoassay was developed with this McAb having a sensitivity of 10 μg/ml and a working range up to 10 μ/ml of OA in standard solutions.
[Show abstract][Hide abstract] ABSTRACT: A monoclonal antibody (McAb) was produced after fusion of mouse (X63.Ag18.104.22.168) myeloma cells with spleen cells isolated from female Balb-c/NZB F1 hybrid mice immunized with aflatoxin B1 (oxine)-keyhole limpet haemocyanin conjugate. The hybridoma cell line producing antibody specific for aflatoxin B1 (AFB1) was grown in tissue culture and as an ascites tumour. The ascitic fluid gave suitably high dilution titres (1:800 000) by enzyme immunoassay and was conjugated to horseradish peroxidase by a two-step procedure with glutaraldehyde. The conjugate was used to develop a direct competitive enzyme-linked immunosorbent (ELISA) assay for AFB1. The sensitivity of the ELISA was 0–2 ng/ml with a working range up to 10 ng/ml for AFB1. The specificity of the McAb was determined and it was shown not to cross-react significantly with any of the metabolites tested. This McAb and the direct competitive ELISA described may prove of use in the detection of AFB1 in foods and feeds.
[Show abstract][Hide abstract] 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 B1, 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile-0.5% KCl-6% H2SO4 (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 acetonitrile 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 HCl 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 less than 12% for B1 and OA but as high as 17% for T2.
Journal - Association of Official Analytical Chemists 73(1):71-6.