Differential induction of sister chromatid exchanges by indirect-acting mutagen-carcinogens at early and late stages of embryonic development

To examine the development of drug-metabolizing enzyme systems in the early chick embryo, the procarcinogens, aflatoxin B₁ (AF-B₁) and 2-acetylamino-fluorene (2-AAF), and the direct-acting carcinogen, ethyl methanesulfonate (EMS) (positive control), were given to embryos; the sister-chromatid-exchange (SCE) technique was used as an indicator of conversion to active mutagenic metabolities. Chick embryos at two stages of incubation (3-day and 6-day) were exposed to the same graded series of dosages of the compounds for a period of 22 hours. All three mutagens increased the frequency of SCE above the control rate of 1.8 SCEs/cell. While a dose-dependent increase in SCE was obtained for both procarcinogens at each age, the mean SCE frequency was significantly higher in the 6-day embryos for each dosage given. In contrast, the direct-acting mutagen, EMS, gave a reduced level of SCEs at the older age. These results suggest that the ability of early chick embryos to activate promutagens to forms capable of inducing SCE increases as development advances from three to six days of incubation (DI). In the 6-day embryo, the metabolic conversion is enhanced, resulting in a significant increase in the mutagenicity of the test chemicals.     

Mutagenicity and toxicity of aflatoxin precursors

The Salmonella/microsome test and the chick embryo test were used to determine the mutagenicity and toxicity of five aflatoxin B₁ precursors. A definite pattern emerges: the nearer to B₁ an intermediate appears in the biosynthetic pathway, the more potent is its mutagenicity and toxicity.     

Effect of Platelet-Activating Factor (PAF) on Preimplantation Mouse B6D2F1/J Embryo Formation

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent signaling phospholipid that has been implicated in a variety of reproductive processes. Human, rabbit, and mouse preimplantation embryos produce and secrete PAR Anti-PAF antibodies interfere with mouse preimplantation development. A controversy exists on whether exogenous PAF is beneficial to preimplantation embryo development. The study objective was to determine the effect of exogenous PAF on embryo formation. One-cell mouse B₆D₂F₁/J embryos were collected from PMSG/hCG primed females mated with fertile males. Embryos were exposed to PAF (0–10 μM) in MEM (0.3% BSA) for 15 min, then cultured in MEM (0.3% BSA) in a 5% CO₂ in air, 95% relative humidity at 37°C atmosphere, for 120 hr to the hatched blastocyst stage. PAF (0.1 or 0.01 μM) significantly (P < 0.05) improved preimplantation embryo development and formation in vitro. PAF at higher doses had no significant effect. Supplementation of culture medium with exogenous PAF was beneficial to preimplantation embryo development in B₆D₂F₁/J mice.     

Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice

Children are generally more sensitive to toxicants than adults, including an increased sensitivity to genotoxic carcinogens. We previously demonstrated that neonatal mice are also more sensitive to the mutagenic effects of the direct alkylating agents N‐ethyl‐N‐nitrosoamine and the arylamine 4‐aminobiphenyl than adult mice. In this study, we have evaluated the effect of age on the mutagenicity of the fungal toxin and liver carcinogen aflatoxin B₁ (AFB₁). Neonatal Big Blue transgenic mice were treated with 6 mg/kg AFB₁, a treatment that produces liver tumors, while adult mice were treated with 6 and 60 mg/kg AFB₁, treatments that do not result in tumors. The cII liver mutant frequency (MF) in mice treated with AFB₁ as neonates was 22‐fold higher than in control neonatal mice, whereas the treatment of adult mice with either dose of AFB₁ did not significantly increase the liver MF over the controls. In AFB₁‐treated neonatal mice, the frequency of G:C → T:A transversion, a major type of mutation induced by AFB₁, was about 82‐fold higher than for the control and 31‐fold higher than for adult mice treated with 60 mg/kg AFB₁. Our mutagenicity findings parallel the relative carcinogenicity of AFB₁ in neonatal and adult mice, and are consistent with previous observations of the lower level of hepatic glutathione S‐transferase and higher level of hepatic AFB₁‐DNA adduction in neonatal mice compared to adult mice. Environ. Mol. Mutagen. 2010. Published 2009 by Wiley‐Liss, Inc.     

Effects of black tea theafulvins on aflatoxin B(1) mutagenesis in the Ames test

Black tea theafulvins, a fraction of thearubigins isolated from black tea aqueous infusions, potentiated the mutagenic activity of the mycotoxin aflatoxin B(1) in the Ames test, in the presence of a hepatic S9 activation system derived from Aroclor 1254-treated rats. In contrast, when the S9 activation system was replaced with isolated microsomes, theafulvins suppressed the mutagenicity of the mycotoxin. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, incorporation of the theafulvins into the activation system reduced the mutagenic activity, whereas if it was added before termination of microsomal activity a potentiation of mutagenic response was observed. In in vitro studies, theafulvins inhibited epoxide hydrolase and glutathione S-transferase activities in a concentration-dependent manner. Finally, the mutagenicity of aflatoxin B(1) was much more pronounced in bacteria that were pre-exposed to theafulvins but from which they were subsequently washed off. It may be inferred from the above studies that the genotoxic synergy between aflatoxin B(1) and black tea theafulvins does not occur during the bioactivation of the carcinogen, but may partly be due to decreased deactivation of the reactive intermediate, aflatoxin B(1) 8,9-oxide, by conjugation with glutathione.     

Expression of human cytochrome P450 1A2 in Escherichia coli: a system for biotransformation and genotoxicity studies of chemical carcinogens

In this study we describe the development of strain BMX100,a new Escherichia coli K12 tester strain, derived from MX100,a strain which was constructed for detection of mutagens andfor mechanistic studies of chemical carcinogens. We demonstratehere that strain BMX100 can be used for stable expression ofhuman CYP1A2 or human CYP1A2 fused to rat liver NADPH cytochromeP450 reductase. Mutagenicity of precarcinogens known to be bioactivatedby CYP1A2, namely 2-aminoanthracene (2-AA), aflatoxin Bl (AFB1)and 2-amino-3-methylimidazo[4,5-f]quinoline (1Q), could be detected.The mutagenic activity of 2-AA using BMX100 expressing CYP1A2alone and in combination with rat CYP reductase was respectively10 and 20 times higher than in BMX100 with the standard metabolicactivation system, rat liver S9 fraction. Furthermore, the mutagenicityof 2-AA could be nullified by a-naphthoflavone, a known inhibitorof CYP1A2. IQ responded equally in BMX100 expressing the CYP1A2-reductasefusion protein as compared with usage of rat liver S9 fraction.Rat liver S9 fraction was much more potent in generating a mutagenicresponse to A FBI in BMX100 than in the strain expressing humanCYP1A2 aloneZ or CYP1A2 fused to rat reductase. The resultsdescribed in this study demonstrate that this new E.coli straincan function as a human CYPlA2-competent prokaryotic mutagenicitytest system and they seem to characterize BMX100 as a strainof interest for studies to identify individual human CYPs involvedin bioactivation and bloinactivation reactions of putative genotoxins. ⁴To whom correspondence should be addressed. Tel: +351 1 3610290; Fax: +351 1 3622018; Email: jose.rueff{at}gene.unl.mailpac.pt     

Protective effects of coumarin and coumarin derivatives against carbon tetrachloride-induced acute hepatotoxicity in rats

The comparison of the antioxidant activity of some coumarins with their molecular structure is well determined. However, the protective function of coumarins with various chemical structures against liver toxicity has not yet been well established. Therefore, the aim of this study was to evaluate the possible cytoprotective properties of coumarin and some coumarin derivatives against CCl₄ (carbon tetrachloride)-induced hepatotoxicity. Coumarin (1,2-benzopyrone) and coumarin derivatives esculetin (6,7-dihydroxycoumarin), scoparone (6,7-dimethoxycoumarin) and 4-methylumbelliferone (7-hyroxy-4-methyl) were examined for their protective effect against CCl₄-induced hepatotoxicity in Male Sprague-Dawley rats. A single toxic dose of CCl₄ (1.25 ml kg⁻¹, orally) produced liver damage in rats, seen histologically as centrilobular necrosis. Administration of CCl₄ increased serum enzyme levels of aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP). Pre-treatment of rats with esculetin (31.15 mg kg⁻¹, orally) and scoparone (35 mg kg⁻¹, orally) significantly prevented CCl₄-induced increase in serum enzymes, whereas 4-methylumbelliferone (35 mg kg⁻¹) and coumarin (30 mg kg⁻¹) had no effect against CCl₄-induced rise in serum enzymes. Morphological findings were consistent with the plasma transaminase observations. Among the coumarin analogs, esculetin, which possesses orthodihydroxy coumarins, showed the strongest protective effect against CCl₄-induced liver damage, followed by scoparone, 4-methylumbelliferone and coumarin, respectively. The results of this study indicate that the chemical structures of coumarins play an important role in the prevention of liver toxicity.     

Pathological and biochemical evaluation of coumarin and chlorophyllin against aflatoxicosis in rat

Aflatoxin contamination of animal diet has adverse effects on animal health and productivity. This study was performed to investigate the effect of using coumarin and/or chlorophyllin in rat diet against aflatoxicosis. Fifty-four rats were assigned into 7 groups (6 rats each). G1 was a negative control. G2 received water with coumarin 0.5%. G3 received water with chlorophyllin 0.5%. G4 received water with coumarin 0.5% and chlorophyllin 0.5%. G5-8 fed aflatoxin B₁ 1000 ppb in diet. Group 6–8 were administered similar treatments as G2-4. The experiment ended after 8 weeks. Random glucose, total lipid, total cholesterol, total triglycerides, total protein, serum ALT, AST, creatinine, and urea were measured. Histopathology of liver, kidney and pancreas and immunohistochemical staining of placental glutathione-S-transferase (GST-P) in liver were performed. The glucose serum level, cholesterol, AST, and ALT were elevated in G5 compared to G6-8. The liver and kidney lesions in G5 included vacuolation and necrosis which subsided in G6-8. The necrosis and inflammatory cells infiltration in the pancreas of G5 were absent in G6-8. GST-P positive hepatocytes were abundant in G5, few in G6 and absent in G7 and G8. In conclusion, the chlorophyllin and coumarin possessed protective and anti-carcinogenic effect against aflatoxicosis in rats.     

Transmission of Immunity to Neisseria Gonorrhoeae from Vaccinated Hens to Embryos

Presently, there is no practical laboratory animal model for the evaluation of experimental gonococcal vaccines. The chimpanzee has been used recently (1), but this animal is expensive and difficult to handle. On the other hand, the. susceptibility of the chick embryo to certain pathogenic microorganisms has provided an excellent model for the study of their virulence (2,3,8,9). In our laboratory, we have confirmed the original observation of Bumgarner et al (4), that Neisseria gonorrhoeae colony types T₁ and T₂ (virulent for man) are significantly more virulent for the chick embryo than T₃ and T₄ (non-virulent for man). However, chick embryo neutralization studies (4,5) have shown the unsuitability of the embryo as an animal model for the protective capacity of vaccines against gonorrhea, since there is only a ten-fold difference between the ability of normal and hyper-immune rabbit sera to protect the embryos against gonococcal challenge. Because of these limitations, we have investigated the ability of embryos obtained from Leghorn hens immunized intravenously with experimental gonococcal vaccines, to withstand a challenge with N. gonorrhoeae     

Immunostimulating effects of protein A in immunosurpressed aflatoxin-intoxicated rats

Aflatoxin B₁, the potent carcinogenic compound produced by the Aspergillus flavus group of fungi on food and feed, induces immunosuppressive effects in rodents. In this communication, we report an immunomodulatory approach to abrogate aflatoxin B₁-induced immunotoxicity in rats using protein A of Staphylococcus aureus Cowan 1. We have earlier demonstrated that protein A can protect the animals from toxicities induced by a number of drugs, chemicals and toxins. In the present study various combinations of aflatoxin B₁ exposure and protein A treatment in animals were used. It was observed that protein A could provide protection to animals from aflatoxin B₁-induced immunotoxicity, as measured by a battery of tests assessing cell-mediated immunity (CMI) profile of the host. Various parameters showing suppression of CMI following aflatoxin B₁ exposure were reverted back towards normalcy in protein A-treated animals. It is concluded that protein A may prove to be a useful agent to protect the host from aflatoxin immunotoxicity, in view of its stimulatory effects on various immune functions even after their initial depression due to aflatoxin B₁.     

Toxigenic fungi and mycotoxin content in poultry feedstuff ingredients

Fifty isolates of Aspergillus flavus as well as 25 isolates of each of A. fumigatus, A. ochraceus, A. terreus and Fusarium spp. isolated from poultry feeds were screened for toxin production. Of these isolates, 20% have been detected to exhibited toxigenic activity. The mycotoxin content was determined in samples of grind maize, cotton-seed cake, fish meal, wheat bran and soybean meal. 80% of maize samples were highly contaminated with aflatoxins (480 μ/kg) and zearalenone (40 μ/kg). Aflatoxins B₁ and B₂ were found in 50 and 30% of cotton-seed cake and fish meal, respectively. With wheat bran, 40% of the samples contained aflatoxin B₁ and zearalenone. Soybean meal appeared to be a poor substrate for mycotoxin formation.     

Treatment with aflatoxin B1 for immortalization of human fibroblasts from Li-Fraumeni patients

Immortalization of normal human fibroblasts is a very rare event. Multiple genes such as p53 and cellular senescence genes are possibly involved in immortalization of human fibroblasts, suggesting that multiple treatments with carcinogens are required for the immortalization. We describe here the procedure for immortalization of human fibroblasts (MDAH 087) from Li-Fraumeni cancer syndrome with a germ-line p53 mutation. The cells were subjected to multiple treatments with aflatoxin B₁ (AFB₁) in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), and 3 of 9 MDAH 087 cell cultures treated 1–3 times with 0.1–1 µg/ml AFB₁ became immortal, defined as continuous growth for over 300 population doublings after the first treatment. However, cultures of human fibroblasts from a normal embryo treated under the same conditions failed to escape senescence. The results indicate that the model of human fibroblasts with a mutated p53 allele exposed to AFB₁ is potentially useful for studying mechanisms of chemically induced immortalization.     

ELISA determination of aflatoxin levels in whole nuts

An indirect, double antibody, microtitration plate ELISA technique was used to assay aflatoxin B, in samples of peanuts, Brazil nuts, almonds, hazelnuts and walnuts. The minimum dilutions of nut extracts required to prevent interferences were of 1 in 10 for almonds; of 1 in 20 for Brazil nuts and peanuts; and of 1 in 50 for hazelnuts and walnuts. The ELISA procedure was validated for aflatoxin B, detection in the shelled nuts referred to above by determining the percentage of this toxin recovered from artificially spiked nut samples. The results confirmed that this technique can be used for aflatoxin B₁ determination in these nuts. The comparative studies undertaken using naturally contaminated peanut samples also led to the conclusion that this ELISA protocol can be recommended as an alternative to the already adopted thin‐layer chromatography (TLC) method. It was also observed that placing peanut samples under a UV light is not an advisable method of ascertaining aflatoxin B₁ contamination of these nuts, as levels of 0.6 μg g^‐1 were not detectable.     

Radioimmunoassay for the envelope glycoprotein of subgroup E avian leukosis-sarcoma viruses.

The envelope glycoprotein of subgroup E avian leukosis viruses (gpE) was purified from Rous-associated virus type 0 (RAV-0) and used in double-antibody competition radioimmunoassays (RIA) and radioimmune precipitations (RIP). When embryo extracts from various inbred lines of chickens were tested, the results of assays for chick helper factor (chf) and RAV-0 were in complete phenotypic agreement with those of RIA for gpE. The expression of genes at the gs and gp loci varied among inbred lines. Normal line 15I₅ embryos contained gpE but not group-specific (gs) antigens, whereas extracts from virus-free lines 15₁ and 6₁ contained both classes of antigen. Line 15_B embryo extracts were negative for both gpE and gs antigen. Soluble gpE was found in sera of all chf-positive chickens from RAV-0-free lines. Sera from adult line 15_B birds were categorized as: (1) positive for RAV-0 but negative for antibody; (2) negative for RAV-0 but positive for gpE and antibody to gpE; and (3) negative for RAV-0, antibody to gpE, and gpE. Evidence of antibody production was completely correlated in RIP of iodinated gpE and serum neutralizations of subgroup E virus. Results of RIP and gpE RIA indicated that half of the sera from nonviremic line 15_B chickens contained antibodies to gpE as well as gpE.     

Natural occurrence of mycotoxins in broad bean (Vicia faba l.) seeds and their effect on Rhizobium-legume symbiosis

Seeds of faba bean cultivar Giza 3 were screened for natural presence of mycotoxins. Eleven out of 100 samples were contaminated. Aflatoxins B₁ and B₂ were found in 7 samples while aflatoxins B₁, B₂, G₁ and G₂ and ochratoxin A were each detected twice in separate samples. Mycotoxins at concentrations of 100 or 200 μg kg^?1 soil significantly decreased nodule number, nodule fresh weight and total nitrogenase activity, leading to reductions in dry matter accumulation and nitrogen yield of the bean. Mycotoxins also suppressed specific nitrogenase activity. NADH-dependent glutamate dehydrogenase (NADH-GDH) as well as glutamate synthase (NADH-GOGAT) activities. In addition, mycotoxins inhibited synthesis of leghaemoglobin, carbohydrated and protein in the nodule cytosol. Of the mycotoxins tested, aflatoxin B₁ was the most toxic. The decline in nitrogenase activity and total N concentration in the plants could be attributed to mycotoxins interference with normal nodule physiology and function.     

High‐resolution MRI analysis of breast cancer xenograft on the chick chorioallantoic membrane

The chick chorioallantoic membrane (CAM) model has been successfully used to study angiogenesis, cancer progression and its pharmacological treatment, tumor pharmacokinetics, and properties of novel nanomaterials. MRI is an attractive technique for non‐invasive and longitudinal monitoring of physiological processes and tumor growth. This study proposes an age‐adapted cooling regime for immobilization of the chick embryo, enabling high‐resolution MRI of the embryo and the CAM tumor xenograft. 64 chick embryos were enrolled in this study. The novel immobilization and imaging protocol was optimized in 29 embryos. From d7 to d18 immobilization of the embryo up to 90 min was achieved by cooling at 4 °C pre‐imaging, with cooling times adapted to age. Its application to tumor growth monitoring was evaluated in 15 embryos after xenotransplantation of human MDA‐MB‐231 breast cancer cells on CAM. Tumor volumes were monitored from d4 to d9 after grafting (d11 to d16 after incubation) applying a T₂‐weighted multislice RARE sequence. At d9 after grafting, the tumors were collected and compared with the MRI‐derived data by histology and weight measurements. Additional imaging methods comprising DWI, T₂ mapping, and the bio‐distribution of contrast agents were tested at d9 after grafting in 20 further embryos. With the adaptive cooling regime, motion artifacts could be completely avoided for up to 90 min scan time, enabling high‐resolution in ovo imaging. Excellent anatomical details could be obtained in the embryo and tumors. Tumor volumes could be quantified over time. The results prove the feasibility of high‐resolution MRI for longitudinal tumor and organ growth monitoring. The suggested method is promising for future applications such as testing tailored and/or targeted treatment strategies, longitudinal monitoring of tumor development, analysis of therapeutic efficacies of drugs, or assessment of tumor pharmacokinetics. The method provides an alternative to animal experimentation. Copyright © 2015 John Wiley & Sons, Ltd.     

A radiometric microassay for ornithine decarboxylase

A simple method for purifying [³H]l-ornithine and incubation conditions suitable for estimatingl-ornithine decarboxylase activity are described. Routine and recycled cation exchange procedures for separating putrescine from ornithine are outlined. Blanks using the routine cation exchange method average approx. 0.04% of the radioactivity contained in the substrate; product recovery is approx. 94%. Thel-ornithine decarboxylase assay is proportional to time for at least 8 h. The relationship between substrate purity and the sensitivity of the cation exchange procedures is assessed. Radiochemical purity is the critical determinant of sensitivity for recycled assays. The cation exchange method is compared with the commonly used CO₂-trapping method. The cation exchange method is more specific and approximately three orders of magnitude more sensitive than the CO₂-trapping method.l-Ornithine decarboxylase activity can be measured reliably in samples of embryonic neural tissue having wet-weights of approx. 1 μgl-ornithine decarboxylase activity in the lumbar spinal cord of the chick embryo decreases 25–30 fold from day 5 to day 18 of embryonic development.A cation exchange procedure for estimatingl-lysine decarboxylase activity is also described. Failure to detectl-lysine decarboxylase activity in the chick embryo lumbar spinal cord is contrasted with the previous report of high cadaverine levels in chick embryos.     

The Use of Tadpoles of Bufo melanostictus (Schneider), Rhacophorus leucomystax maculatus (Gray) and Uperodon sp. in the Bioassay of Aflatoxins

Aflatoxins have been found to be lethal to tadpoles of Bufo melanostictus (Schneider), Rhacophorus leucomystax maculatus (Gray) and Uperodon sp. Pathological changes consisting of cytoplasmic vacuolation, enlargement and increased vesicularity of nucleii, and foci of necrosis are produced in the liver and kidney of aflatoxin treated larvae. A method is described for the quantitation of aflatoxin based on the lethal response of the tadpoles. The method is cheap, easily handled and provides reproducible and accurate results; the duration of the test is 4 days. The mean LD₅₀ of aflatoxin B₁ on 15 mm. Bufo larvae, 20 mm. Rhacophorus and 15 mm. Uperodon larvae were approximately 2·8, 1·6 and 0·5 μg./ml. respectively while the LD₅₀ of aflatoxin G₁ on 15 mm. Bufo and 20 mm. Rhacophorus larvae were approximately 12·2 and 9·3 μg./ml. respectively. The sensitivity to aflatoxin decreased with increase of size of the larvae. Retardation of growth and delay in metamorphosis have been observed in aflatoxin treated larvae.The applicability of this method to the assay of aflatoxins in crude extracts of foodstuffs is being investigated.     

Secretion pathway of liver IGF-1 via JAK2/STAT3 in chick embryo under the monochromatic light

This study reveals mechanism of monochromatic light on the IGF-1 secretion of chick embryo liver. The chick embryos were incubated and exposed to continuous red, green, blue light or a dark environment. Compared to other light-treated groups, green light increased IGF-1 and melatonin concentrations both in plasma and liver, and Mel1a, Mel1b and Mel1c receptors expressions in liver but decreased p-JAK2, p-STAT3 and ROS in liver. IGF-1 had a positive correlation with melatonin, but a negative relevance with p-JAK2 and p-STAT3. In vitro, the IGF-1 level in the hepatocyte supernatant was enhanced by melatonin with lower p-JAK2/p-STAT3 and ROS levels, which was suppressed by Mel1c antagonist but not Mel1a/Mel1b or Mel1b antagonists. AG490 (JAK/STAT inhibitor) promoted role of melatonin-Mel1c modulated IGF-1 secretion. These results suggest the antioxidant effect of melatonin mediated the green light-enhanced IGF-1 secretion of chick embryo liver through Mel1c receptor to inhibit the JAK2/STAT3 pathway.     

Production and characterization of antibodies against aflatoxin in laying hens

Polyclonal antibodies against aflatoxins were obtained from egg yolks of laying hens immunized with either aflatoxin B₁ (AFB₁) or aflatoxin M₁ (AFM₁) conjugated to bovine serum albumin (BSA). An indirect enzyme‐linked immunosorbent assay (ELISA) involving the use of AFB₁‐BSA or AFM₁‐BSA conjugate and anti‐chicken IgG‐horseradish peroxidase (HRP) conjugate, was developed for monitoring antibody titers and aflatoxin analysis. Production of the antibody in hens started as early as 10 days after immunization and reached a maximum in 20 days. Competitive indirect ELISA revealed that the antibodies were most specific for AFB₁. They were cross‐reactive with other aflatoxins in the following order: B₁ (100%)>G₁ (22–28%)>B₂ (6–16%)>M₁(3–9%)>G₂ (1–4%). Anti‐AFM₁‐BSA antibodies showed a similar pattern of cross‐reaction to the anti‐AFB₁‐BSA antibodies when AFB₁‐BSA was coated to the ELISA plate. The specificity of anti‐AFM₁‐BSA to AFM₁ was demonstrated when AFM₁‐BSA was coated to the ELISA plate. Cross‐reactivity with different aflatoxins was in the following order: M₁ (531%)>B₁ (100%)>G₂ (41%)>G₁ (19%)>B₂ (3%). The sensitivity for AFB₁ analysis in the indirect ELISA was 0.05–5 ng AFB₁/assay.