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.     

Production of antibody against aflatoxin B1.

Antibody against aflatoxin B1 was obtained after one multiple-site injection of bovine serum albumin-aflatoxin B1 conjugate into rabbits. The antibody has greatest binding efficiency for aflatoxin B1, less efficiency for B2, G1, and Q1, and least for aflatoxicol, G2, and M1. Sterigmatocystin, coumarin, and 4-hydroxycoumarin did not give a cross-reaction with the antibody. The sensitivity of the binding assay for detection of aflatoxin B1 is in the range of 0.2 to 2.0 ng per 0.5-ml sample. Detailed methods for the preparation of the conjugate, production of immune serum, and methods for antibody titer determination are described.     

Enzyme-linked immunosorbent analysis for aflatoxin B1.

An enzyme-linked immunosorbent analysis (ELISA) permitted the detection of less than 10 pg of aflatoxin B1 per ml. The antitoxin was most specific for aflatoxins B1 and B2alpha, and least specific for aflatoxin G1.     

Enzyme-linked immunosorbent analysis for aflatoxin B1.

An enzyme-linked immunosorbent analysis (ELISA) permitted the detection of less than 10 pg of aflatoxin B1 per ml. The antitoxin was most specific for aflatoxins B1 and B2alpha, and least specific for aflatoxin G1.     

Simultaneous occurrence of deoxynivalenol, zearalenone, and aflatoxin in 1982 scabby wheat from the midwestern United States.

Thirty-three samples of wheat of the 1982 crop year from Kansas and Nebraska were analyzed for deoxynivalenol, T-2 toxin, zearalenone, and aflatoxin. Deoxynivalenol was identified in 31 of 33 samples, zearalenone was identified in 3 of 33 samples, and aflatoxin B1 was identified in 23 of 31 samples. One 1982 wheat sample from Illinois and one from Texas were also contaminated with deoxynivalenol at 1,200 and 600 ng/g, respectively. None of the samples contained detectable T-2 toxin. The mean concentration of deoxynivalenol was 1,782 +/- 262 ng/g, and the concentrations of aflatoxin B1 ranged from 0.8 to 17.0 ng/g, with a mean of 3.37 +/- 0.7. Zearalenone concentrations of the three positive samples were 35, 90, and 115 ng/g. However, density segregation of two other samples which tested negative yielded light fractions, comprising less than 2% of the samples, contaminated at 230 and 254 ng of zearalenone per g; calculated zearalenone concentrations for these two samples were below the limit of detection of the method. The high frequency of aflatoxin B1 and deoxynivalenol in wheat from the 1982 crop is unprecedented, as is the simultaneous contamination of some samples with deoxynivalenol, zearalenone, and aflatoxin B1.     

Simultaneous occurrence of deoxynivalenol, zearalenone, and aflatoxin in 1982 scabby wheat from the midwestern United States

Thirty-three samples of wheat of the 1982 crop year from Kansas and Nebraska were analyzed for deoxynivalenol, T-2 toxin, zearalenone, and aflatoxin. Deoxynivalenol was identified in 31 of 33 samples, zearalenone was identified in 3 of 33 samples, and aflatoxin B1 was identified in 23 of 31 samples. One 1982 wheat sample from Illinois and one from Texas were also contaminated with deoxynivalenol at 1,200 and 600 ng/g, respectively. None of the samples contained detectable T-2 toxin. The mean concentration of deoxynivalenol was 1,782 +/- 262 ng/g, and the concentrations of aflatoxin B1 ranged from 0.8 to 17.0 ng/g, with a mean of 3.37 +/- 0.7. Zearalenone concentrations of the three positive samples were 35, 90, and 115 ng/g. However, density segregation of two other samples which tested negative yielded light fractions, comprising less than 2% of the samples, contaminated at 230 and 254 ng of zearalenone per g; calculated zearalenone concentrations for these two samples were below the limit of detection of the method. The high frequency of aflatoxin B1 and deoxynivalenol in wheat from the 1982 crop is unprecedented, as is the simultaneous contamination of some samples with deoxynivalenol, zearalenone, and aflatoxin B1.     

Sensor system based on molecular-imprinted polymer membranes for the selective recognition of aflatoxin B1

lymer membranes, synthesized using acrylamide as a functional monomer, were characterized by sufficient mechanical stability and high adsorbtion capability towards aflatoxin B1. The molecularly-imprinted polymer membranes were characterized by the pronounced imprinting effect as well as by insignificant adsorbtion of aflatoxins B2 and G2. Therefore, the synthetic analogues of bioreceptors able to individual recognition of aflatoxin B1 were obtained and used as a basis for the optical sensor system for aflatoxin B1 detection in a concentration range 1-500 ng/ml.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Development and validation of a method for the quantitative determination of aflatoxin contaminants in Maytenus ilicifolia by HPLC with fluorescence detection

A method for the quantification of aflatoxins B1, G1, B2 and G2 in the medicinal herb Maytenus ilicifolia was developed and validated. The method used immunoaffinity columns for sample clean-up and HPLC with fluorescence detection without any derivatisation step. The method showed good inter-day accuracy (bias values in the range 4.5-10.7%) and precision (5-16% RSD) when applied to the determination of levels of aflatoxins ranging from 7 to 20 ppb in the plant material. The detection limits for samples of the plant material spiked with aflatoxins were 3.5 ng/g for B1 and G1 and 0.1 ng/g for B2 and G2. The method was successfully applied to commercial samples of Maytenus ilicifolia for the screening of aflatoxin contaminants.     

Influence of temperature cycling on the production of aflatoxins B1 and G1 by Aspergillus parasiticus.

The effect of temperature cycling on the relative productions of aflatoxins B1 and G1 by Aspergillus parasiticus NRRL 2999 was studied. The cycling of temperature between 33 and 15 degrees C favored aflatoxin B1 accumulation, whereas cycling between 35 and 15 degrees C favored aflatoxin G1 production. Cultures subjected to temperature cycling between 33 and 25 degrees C at various time intervals changed the relative productions of aflatoxins B1 and G1 drastically. Results obtained with temperature cycling and yeast extract-sucrose medium with ethoxyquin to decrease aflatoxin G1 production suggest that the enzyme system responsible for the conversion of aflatoxin B1 to G1 might be more efficient at 25 degrees C than at 33 degrees C. The possible explanation of the effect of both constant and cycling temperatures on the relative accumulations of aflatoxins B1 and G2 might be through the control of the above enzyme system. The study also showed that greater than 57% of aflatoxin B1, greater than 47% of aflatoxin G1, and greater than 50% of total aflatoxins (B1 plus G1) were in the mycelium by day 10 under both constant and cyclic temperature conditions.     

Production of Aflatoxin on Wheat and Oats: Measurement with a Recording Densitometer

A method has been developed for the production of aflatoxin by growing Aspergillus flavus NRRL 3145 on solid substrate wheat. Optimal yields of 900 mug of aflatoxin G(1) and 900 mug of aflatoxin B(1) per g of substrate were obtained in 4 to 5 days at 28 C. A study of aflatoxin production on hulls and groats of oats and on whole oats by A. flavus strains NRRL 2999, NRRL 3000, and NRRL 3145 revealed that aflatoxin was produced on all three substrates, although production was very slight on hulls. Strain NRRL 3145 grown on solid substrate groats produced the largest amounts of aflatoxin: 580 mug of B(1) and 450 mug of G(1) per g of substrate. A densitometric method for reading thin-layer chromatographic plates is described; this is more objective and more accurate than the visual methods previously used for the determination of all four aflatoxins.     

Production and characterization of monoclonal antibodies for aflatoxin B1

Hybridomas that secreted antibodies for aflatoxin B1 were selected using two immunization protocols referred to as A and B. Protocol A is a standard immunization method and resulted in the selection of only two clones that produced monoclonal antibodies against aflatoxin B1. In protocol B a unique immunization schedule which resulted in the generation of 10 hybridomas is described. Of the 10, one antibody was highly specific to B1, four antibodies reacted equally strongly with B1, G1 and weakly with B2. Another four reacted strongly with B1 and weakly with B2 and G1. One clone reacted equally strongly with B1, G1 and B2. Interestingly all the 10 antibodies showed little or no cross-reaction with G2.     

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.     

Incidence of aflatoxins and aflatoxin producing fungi in animal feedstuffs

Fifty four samples including 5 of broken rice, 8 of corn grains, 8 of corn gluten feed, 13 of cottonseed cake and 4 each of rice polish, corn gluten, sesame oil cake, guar meal and wheat bran were screened for the presence of aflatoxins. Among all the samples, 14 were damaged and 40 apparently undamaged. The incidende of aflatoxins was found to be 60, 25, 25, and 23 per cent in broken rice, corn grains, corn gluten feed and cottonseed cake. Aflatoxins were not detected from rice polish, corn gluten, sesame oil cake, guar meal and wheat bran. Damaged sample revealed a much higher incidence i.e. 50 per cent as compared to undamaged ones i.e. 7.5 per cent. Mean concentration of aflatoxin B and G was found to be 15.5 and 12.2 ppb respectively.Cultural examination of aflatoxin positive feedstuffs yielded 39 isolates of different fungi including 21 of Aspergillus, 7 of Mucor, 6 of Rhizopus, 4 of Fusarium and one of Penicillium. These strains when tested for aflatoxin producing ability, revealed this property in only one isolate, identified as Aspergillus parasiticus.     

Aflatoxin Production in Meats. I. Stored Meats

Aflatoxins were produced on fresh beef (in which bacterial spoilage was delayed with antibiotics), ham, and bacon inoculated with toxinogenic fungi and stored at 15, 20 and 30 C. Meats stored at 10 C were spoiled by bacteria and yeast before detectable levels of aflatoxins were produced. High levels of aflatoxins were formed in meats stored at 20 C; one sample supported the production of 630 mug of aflatoxins per g of meat, the major portion (580 mug) of which was aflatoxin G(1). Meats stored below 30 C developed higher levels of aflatoxin G(1) than B(1), but at 30 C Aspergillus flavus produced equal amounts of B(1) and G(1), whereas A. parasiticus continued to produce more G(1) than B(1).     

Production and characterization of aflatoxin B2a antiserum.

The specificity and sensitivity of antiserum elicited from rabbits against aflatoxin B2a-bovine serum albumin conjugates were characterized with a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA). Aflatoxin B1 was first converted to aflatoxin B2a and then conjugated to bovine serum albumin and horseradish peroxidase by a reductive alkylation method. The antiserum was developed in New Zealand white rabbits by multiple-site injection with the aflatoxin B2a-bovine serum albumin conjugate. Antibody titers were determined by both RIA and ELISA. Competitive RIAs with various aflatoxin analogs indicated that the antiserum was most reactive with aflatoxin B1 and slightly cross-reactive with aflatoxins B2a, B2, and M1. Competitive ELISAs showed the antiserum to be equally specific for aflatoxins B2a and B12 and less reactive with aflatoxins B2 and M1. The relative sensitivities of RIA and ELISA for aflatoxin B1 quantitation were 100 and 10 pg per assay, respectively.     

Development of immunochromatography strip-test using nanocolloidal gold-antibody probe for the rapid detection of aflatoxin B1 in grain and feed samples

An immunochromatography (ICG) strip test using a nanocolloidal gold-antibody probe was developed and optimized for the rapid detection of aflatoxin B1 (AFB1). A monoclonal antibody specific to AFB1 was produced from the cloned hybridoma cell (AF78), coupled with nanocolloidal gold, and distributed on the conjugate pad of the ICG strip test. The visual detection limit of the ICG strip test was 0.5 ng/ml, and this method showed a cross-reaction to aflatoxin B2, G1, and G2. In total, 172 grain and feed samples were collected and analyzed by both the ICG strip test and HPLC. The results of the ICG strip test showed a good agreement with those obtained by HPLC. These results indicated that the ICG strip test has a potential use as a rapid and cost-effective screening tool for the determination of AFB1 in real samples and could be applied to the preliminary screening of mycotoxin in food and agricultural products, generating results within 15 min without complicated steps.     

Incidence of aflatoxin producing strains and aflatoxin contamination in dry fruit slices of quinces (Cydonia oblonga Mill.) from the Indian state of Jammu and Kashmir

An investigation was undertaken to obtain data on the occurrence of aflatoxins and the aflatoxin producing potential of Aspergillus flavus strains isolated from dry fruit slices of quinces produced in jammu and Kashmir, India. A total of 147 A. flavus isolates recovered from dr fruit slices were grown in liquid rice flour medium and screened for the production of various aflatoxins by thin layer chromatography. The results showed that 23.14% of the tested isolates were aflatoxigenic, producing aflatoxins B1 and B2 in varying amounts. Aflatoxins G1 and G2 were not detected. All 25 of the investigated market samples were also found to be aflatoxin B1 positive and the level of contamination ranged from 96 to 8164 micrograms/kg of the dry fruit which is quite high in comparison to the permissible level of 30 ppb. As per these results biochemical composition of dry fruit slices of quinces, along with climatic conditions seem to be very favourable for aflatoxin production by the toxigenic A. flavus strains. Therefore, monitoring of aflatoxins in dry fruit slices of quinces is recommended for this region.     

Aflatoxins of Aspergillus flavus Grown on Cassava

Aspergillus flavus Link ex Fries was grown on cassava (Manihot utilissima) and Czapek-Dox media at 31±1°C and 90 per cent relative humidity for 8 weeks. Isolation and purification of the toxic and carcinogenic metabolic products of the mould by paper and thin layer chromatography are described. High concentrations of aflatoxin B1, B2, G1 and G2 were synthesized by the mould grown on cassava for 4 weeks. In addition to these four major toxic components, 9 other fluorescent materials were observed on the chromatograms of crude products from cassava. It is concluded that the high moisture, high polysaccharide and low nitrogen content of cassava constitute a favourable nutritional condition at high temperatures and high relative humidity for the growth of A. flavus and the synthesis of relatively high concentrations of aflatoxin. This conld represent a serious health hazard in the moist tropics.     

Sodium bicarbonate reduces viability and alters aflatoxin distribution of Aspergillus parasiticus in Czapek's agar

The potential of sodium bicarbonate to inhibit growth of and aflatoxin synthesis by Aspergillus parasiticus was examined in Czapek's agar (CA), a medium in which fluorescence under UV light indicates aflatoxin production. Incorporation of sodium bicarbonate (SB) into CA at 0.011, 0.022, and 0.033 mol% reduced cell viability 63-, 10(3)-, and greater than 10(7)-fold, respectively. Colonies resulting from surviving cells did not fluoresce under UV light, but thin-layer chromatography analysis of culture extracts detected aflatoxins. Potassium bicarbonate (KB) at 0.011 and 0.022 mol% produced inhibitory effects similar to those of SB, but NaCl and silica had no effect. After 7 days, control cultures had the normal aflatoxin distribution (B1 greater than G1 greater than B2 greater than G2), but this distribution shifted to B2 greater than B1 approximately equal to G2 greater than G1 during prolonged incubation. Cultures supplemented with SB and KB contained mostly aflatoxins B1 and G1 after 28 days. Both SB and KB raised the pH of CA to 7.5 to 8.5 at the time of growth. Culture growth on CA adjusted to pH 7.5 to 8.5 with NaOH was not inhibited but exhibited reduced fluorescence and elevated levels of aflatoxins B1 and G1. Thus, while bicarbonate inhibition of growth could not be attributed to pH elevation, the lack of culture fluorescence on CA-SB and CA-KB and the altered aflatoxin distribution were caused by the ability of SB and KB to elevate pH.