An enzyme immunoassay system for aflatoxin B1

Indirect enzyme immunoassay based on immobilized conjugate of aflatoxin B₁ carboxymethyloxime with bovine serum albumin and polyclonal rabbit antibodies allows determining aflatoxin B₁ with a low relative cross-reactivity against aflatoxin B₂, G₁, G₂, M₁ B_2a, and G_2a and sterigmatocystin (15.5, 15.5, 1.7, 1.0, 0.03, 0.03 and 0.01%, respectively) with a sensitivity of 0.04 ng per well or 4.0 ng per ml organic solvent.     

In situ absorption of aflatoxins in rat small intestine

To evaluate the rate at which the four main aflatoxins (aflatoxins B₁, B₂, G₁ and G₂) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (k _a ) of 5.84±0.05 (aflatoxin B₁), 4.06±0.09 (aflatoxin B₂), 2.09±0.03 (aflatoxin G₁) and 1.58±0.04 (aflatoxin G₂) h^–1, respectively.     

Comparative immunochemical characterization of polyclonal and monoclonal antibodies to aflatoxin B1

Four hybrid clones (MM-(AB₁)-1, MM-(AB₁)-2, MM-(AB₁)-3, and MM-(AB₁)-4) were obtained by hybridoma technology involving the immunization of BALB/c mice with a BSA conjugate of aflatoxin B₁ carboxymethyloxime derivative. Antibodies produced by these clones varied in their ability to recognize the aflatoxin B₁ analogues. The sensitivity of enzyme immunoassay based on all monoclonal antibodies was higher compared to analysis based on polyclonal rabbit antibodies (0.1 and 0.4 ng/ml, respectively).     

The analysis of aflatoxins by high-performance liquid chromatography.

The potential of high-performance liquid chromatography as a technique for separating aflatoxins B₁ B₂, G₁, G₂, B_2a, Q₁, M₁, P₁, aflatoxicol, and a degradation product of aflatoxin B₁, 2,3-dihydrodiol, has been assessed. A microparticulate silica adsorption column used with a 1:1 chloroform -dichloromethane eluant provided good resolution of aflatoxins B₁, B₂, G₁, and G₂ but the addition of 1% propan-2-ol was necessary for the elution of aflatoxins M₁ and Q₁. By selecting appropriate solvent mixtures, good resolution of all of the aflatoxins studied was obtained using columns containing an octadecyl (C₁₈) reversed-phase bonded to a microparticulate support. Details are given for resolving: (1) aflatoxins B₁, B₂, G₁, and G₂ using a 5% tetrahydrofuran-15% dimethylformamide in water eluant and (2) aflatoxins B₁ B_2a, Q₁ M₁ P₁ aflatoxicol, and a product of aflatoxin B₁ 2,3-dihydrodiol treated with Tris-buffer, using either 15% dimethylformamide in water or 10% tetrahydrofuran in water as eluant.     

The influence of the transpiration rate on uptake and transport of potassium ions in barley plants

Summary The vegetation points of branches of Caralluma frerei (Asclepiadaceae) were treated with 300, 100 and 30 ppm of crude aflatoxin (36% B₁, 38% G₁, 3% B₂, 2% G₂) and with toxin-free control. Application of 300 and 100 ppm aflatoxin resulted in stop of growth and death of the upper leaves and flower buds. Malformations or wilting was not observed in any case. Branches treated with 30 ppm aflatoxin and with control solution developed normally. It is concluced that under the experimental conditions used aflatoxin has an unspecific toxic effect.     

Production and characterization of polyclonal and monoclonal antibodies against Aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay

From a single aflatoxin B₁ oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B₁, B₂, G₁ and G₂; B₁ and B₂; B₁ and G₁; and G₁ alone. No antibody preparations reacted with aflatoxin M₁. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.     

The production of aflatoxin B1 or G1 by Aspergillus parasiticus at various combinations of temperature and water activity is related to the ratio of aflS to aflR expression

The influence of varying combinations of water activity (a_w) and temperature on growth, aflatoxin biosynthesis and aflR/aflS expression of Aspergillus parasiticus was analysed in the ranges 17–42°C and 0.90–0.99 a_w. Optimum growth was at 35°C. At each temperature studied, growth increased from 0.90 to 0.99 a_w. Temperatures of 17 and 42°C only supported marginal growth. The external conditions had a differential effect on aflatoxin B₁ or G₁ biosynthesis. The temperature optima of aflatoxin B₁ and G₁ were not at the temperature which supported optimal growth (35°C) but either below (aflatoxin G₁, 20–30°C) or above (aflatoxin B₁, 37°C). Interestingly, the expression of the two regulatory genes aflR and aflS showed an expression profile which corresponded to the biosynthesis profile of either B₁ (aflR) or G₁ (aflS). The ratios of the expression data between aflS:aflR were calculated. High ratios at a range between 17 and 30°C corresponded with the production profile of aflatoxin G₁ biosynthesis. A low ratio was observed at >30°C, which was related to aflatoxin B₁ biosynthesis. The results revealed that the temperature was the key parameter for aflatoxin B₁, whereas it was water activity for G₁ biosynthesis. These differences in regulation may be attributed to variable conditions of the ecological niche in which these species occur.     

Products of Spontaneous Conjugation of Aflatoxins with Bovine Serum Albumin: Immunochemical Properties

Products of spontaneous conjugation of aflatoxins B₁, G₁, and G₂ with bovine serum albumin (BSA) were shown to interact with antibodies against aflatoxins. Solid-phase BSA conjugates inhibited the binding of aflatoxins by antiaflatoxin antibodies. Antisera against BSA–B₁, BSA–G₁, and BSA–G₂ were obtained and their specificity determined. The mechanisms of spontaneous binding of aflatoxins by proteins are discussed.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

Specific features of albumin interactions with hemiacetals of aflatoxins and sterigmatocystin

Aflatoxin B2a (AB2a), aflatoxin G2a (AG2a), and hemiacetal of sterigmatocystin have been shown to form immunoreactive conjugates with albumin. The conjugates were formed following incubation of solution mixtures at room temperature for 1 h, as demonstrated by spectrophotometry and enzyme immunoassay. Anti-AB2a antibodies reacted with AB2a, aflatoxin B1, and aflatoxin AB2 (100, 8.8, and 5.9%, respectively); a similar result was obtained for anti-AG2a antibodies reacting with AG2a, aflatoxin G1, and aflatoxin AG@2 (100, 2.5, and < 1.0%, respectively). Binding of anti-AB2a and anti-AG2a antibodies to solid-phase conjugates of AB2a or AG2a exhibited similar analytical characteristics.     

Reduction of aflatoxins by Rhizopus oryzae and Trichoderma reesei

This study evaluated the ability of the microorganisms Rhizopus oryzae (CCT7560) and Trichoderma reesei (QM9414), producers of generally recognized as safe (GRAS) enzymes, to reduce the level of aflatoxins B₁, B₂, G₁, G₂, and M₁. The variables considered to the screening were the initial number of spores in the inoculum and the culture time. The culture was conducted in contaminated 4 % potato dextrose agar (PDA) medium, and the residual mycotoxins were determined every 24 h by HPLC-FL. The fungus R. oryzae has reduced aflatoxins B₁, B₂, and G₁ in the 96 h and aflatoxins M₁ and G₂ in the range of 120 h of culture by approximately 100 %. The fungus T. reesei has reduced aflatoxins B₁, B₂, and M₁ in the 96 h and aflatoxin G₁ in the range of 120 h of culture by approximately 100 %. The highest reduction occurred in the middle of R. oryzae culture.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.     

Inhibitory effect of hena (Lawsonia inermis leaves) and carrot root on aflatoxin production byAspergillus parasiticus

Experiments were undertaken to evaluate the effect of some natural products (hena, and carrot root) on growth and aflatoxins production byAspergillus parasiticus FRR 2752. Powdered hena (0.5 and 5%) inhibited mycelial growth and delayed 1 sporulation ofA parasiticus during 7 days. The inhibition of growth was increased with increasing the added amount. Aflatoxins production byA parasiticus was reduced with 40–100% in the presence of hena (Lawsonia inermis leaves). Carrot root extract stimulated the fungal growth and aflatoxin production, whereas carrot root fibers slightly enriched fungal growth, inhibited aflatoxins production (B₁, G₁, and G₂), but there was no inhibition of aflatoxin B₂ production byA parasiticus.     

Mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds for human consumption

A survey was carried out to obtain data on the occurrence of mycotoxins and the mycotoxin-producing potential of fungi isolated from nuts (almonds, peanuts, hazelnuts, pistachio nuts) and sunflower seeds in Spain. Thin-layer chromatography was used to separate the toxins. Aflatoxins were detected in one sample of almonds (95 ppb aflatoxin B₁ and 15 ppb aflaxtoxin B₂) and in one sample of peanuts at a level below 10 ppb of aflatoxin B1. 100% of samples showed variable incidence of fungal contamination. The predominant fungi present in samples were Penicillium spp, Aspergillus niger, A. flavus, A. glaucus and Rhizopus spp. The results showed that isolates of different species were able to produce aflatoxins B₁, B₂, G₁, and G₂, sterigmatocystin, ochratoxin A, patulin, citrinin, penicillic acid, zearalenone, and griseofulvin.     

Liquid Chromatography of Aflatoxins

There are numerous reports on studies of aflatoxins, but there are only a few reports on the isolation and mutual separation of aflatoxins by liquid chromatography. Following the previous reports on the liquid chromatography of four kinds (B₁, B₂, G₁ and G₂) of aflatoxins, the authors carried out the chromatography of six kinds of aflatoxins including aflatoxins B_2a and G_2a. Various kinds of adsorbents and eluting solvents were examined, and then the good mutual separation of aflatoxins was obtained by using the Sephadex G-10 (CM), a newly prepared adsorbent containing carboxymethyl group, and pure water for eluting solvent. Aflatoxins G_2a, B_2a, G₂, B₂, G₁ and B₁ were eluted in this order.     

Rapid On-Site Sensing Aflatoxin B1 in Food and Feed via a Chromatographic Time-Resolved Fluoroimmunoassay

Aflatoxin B₁ poses grave threats to food and feed safety due to its strong carcinogenesis and toxicity, thus requiring ultrasensitive rapid on-site determination. Herein, a portable immunosensor based on chromatographic time-resolved fluoroimmunoassay was developed for sensitive and on-site determination of aflatoxin B₁ in food and feed samples. Chromatographic time-resolved fluoroimmunoassay offered a magnified positive signal and low signal-to-noise ratio in time-resolved mode due to the absence of noise interference caused by excitation light sources. Compared with the immunosensing performance in previous studies, this platform demonstrated a wider dynamic range of 0.2-60 μg/kg, lower limit of detection from 0.06 to 0.12 µg/kg, and considerable recovery from 80.5% to 116.7% for different food and feed sample matrices. It was found to be little cross-reactivity with other aflatoxins (B₂, G₁, G₂, and M₁). In the case of determination of aflatoxin B₁ in peanuts, corn, soy sauce, vegetable oil, and mouse feed, excellent agreement was found when compared with aflatoxin B₁ determination via the conversational high-performance liquid chromatography method. The chromatographic time-resolved fluoroimmunoassay affords a powerful alternative for rapid on-site determination of aflatoxin B₁ and holds a promise for food safety in consideration of practical food safety and environmental monitoring.     

Aflatoxin-producing Potential of Isolates of the Aspergillus flavus-oryzae Group from Peanuts (Arachis hypogaea)

Seventy-eight samples of farmer stock peanuts, representing peanuts grown in nine different geographical areas during 1964, were assayed for aflatoxin and examined for associated microflora. Only two samples contained more than 50 ppb of aflatoxin. Infestation by members of the Aspergillus flavus-oryzae group varied from 35 to 100% of the kernels per area and from 1 to 100% of the kernels per sample. Aflatoxin production by individual isolates ranged from 0 to 349,143 ppb under the test conditions employed. In general, the isolates produced 8 to 10 times more B₁ than B₂, and no isolate producing aflatoxins G₁ or G₂ was found. The importance of proper postharvest handling of peanuts is emphasized by the prevalence of isolates of A. flavus-oryzae capable of producing aflatoxins on farmers stock peanuts.     

Divergence of West African and North American Communities of Aspergillus Section Flavi

West African Aspergillus flavus S isolates differed from North American isolates. Both produced aflatoxin B₁. However, 40 and 100% of West African isolates also produced aflatoxin G₁ in NH₄ medium and urea medium, respectively. No North American S strain isolate produced aflatoxin G₁. This geographical and physiological divergence may influence aflatoxin management.     

Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues

The luncheon meat samples analyzed, which were produced locally by the two main luncheon meat producing companies in Egypt were relatively highly contaminated either by moulds and yeasts in general, aflatoxigenic species and aflatoxin residues in particular. The most frequently encountered fungi from the samples were yeasts, Aspergillus niger, A. flavus, Penicillium chrysogenum, Rhizopus stolonifer, Mucor circinelloides. Less common were Cladosporium sphaerospermum, Alternaria alternata, Mycosphaerella tassiana, P. aurantiogriseum and P. oxalicum. The most important aflatoxigenic species, A. flavus, was isolated frequently. It was 10% of the total fungal isolates from both samples of the two companies. Seven luncheon meat samples out of 50 analyzed were positive for aflatoxin B₁ or B₁ and G₁, while all samples were negative for aflatoxins B₂, G₂, M₁ and M₂. Aflatoxin B₁ was detected only in 4 and 3 samples out of 25 analyzed from each of company A and B, respectively. The highest detectable level, 11.1 ppb, was recorded in a sample from company B and the least, 0.5 ppb, in a sample from company A. Aflatoxin G₁, at concentration of 3.2 ppb, was detected in only one sample of the aflatoxin B₁ – contaminated 3 samples of company B: this sample also had the highest level of aflatoxin B₁. Some luncheon meat samples had higher numbers of aflatoxigenic A. flavus than others, however these samples were negative for aflatoxins. The hazardous potential of such contamination will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

An Ultra-Sensitive Monoclonal Antibody-Based Competitive Enzyme Immunoassay for Sterigmatocystin in Cereal and Oil Products

Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B₁, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B₁, B₂, G₁, G₂, and M₁. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg·mL⁻¹) ELISA format, in which the antibody was diluted to 1∶80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng·g⁻¹ for wheat, 0.06 ng·g⁻¹ for maize, and 0.1 ng·g⁻¹ for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90–104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products.