Competitive Elisa for quantifying small Amounts of aflatoxin B1

An indirect ELISA was developed to quantify aflatoxin B₁ (AFB₁). The detection limit was 0.025 ng ml^‐1. The test used polyvinyl chloride (PVC) plates, activated with AFB₁ bound to bovine serum albumin (BSA). Polyclonal antibodies were raised in rabbits against AFB₁‐BSA. The specific anti‐AFB₁antibodies were recovered from the crude antiserum by affinity chromatography from a column containing immobilized BSA on Nylon 6–6. Goat anti‐rabbit IgG antibodies bound to peroxidase were used to detect the rabbit IgG anti‐AFB₁ antibodies bound to PVC plates. The colour developed by the subsequent enzyme conversion of the substrate was detected by spectrophotometry. The developed colour gave clear absorbance differences at varying doses of AFB₁. Cross‐reactivity with aflatoxin B₂, aflatoxin G₁ and aflatoxin G₂ was measured, showing percentages of 5.43, 64.5 and 5.07 respectively.     

Application of an automated particle‐based immunosensor for the detection of aflatoxin B1 in foods

A method using an automated particle immunosensor is described which can easily detect aflatoxin B₁ down to a level of 4 ng g^‐1 (4 ppb) in reference food materials. The assay takes approximately 8 min and employs a simple extraction procedure which takes <60 min to complete. Experiments with other aflatoxins indicate that only G₂ shows significant cross‐reactivity (23%). The potential of the method for both surveillance and application in the food industry is assessed.     

Survey of aflatoxin B1 and ochratoxin A in commercial green coffee beans by high‐performance liquid chromatography linked with immunoaffinity chromatography

The natural occurrence of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) has been surveyed in 47 samples of commercial green coffee beans. Samples were collected in Nagoya City, Japan, during 1988–93. Using a combination of immunoaffinity chromatography and high‐performance liquid chromatography analysis, AFB₁ and OTA were quantified with detection limits of 2 ng kg^‐1 and 0.1 μg kg^‐1 respectively. Positive rates and levels of AFB₁and OTA were 32% and 2.0–32.9 ng kg^‐1, and 30% and 0.1–17.4 μg kg^‐1 respectively. The samples contaminated with AFB₁ or OTA were mainly imported from African and Asian countries, and several samples were found to be positive for these two mycotoxins for the first time in Japan, although their levels were low.     

Synthesis and characterization of three Aflatoxin B1‐protein conjugates

Pure aflatoxin B, was obtained after extraction and chromatographic purification from cultures of A. flavus. Aflatoxin B, derivatives suitable for conjugate formation were synthesized. Intermediates and conjugates were characterized spectrophotometrically.     

Effect of aflatoxin B1 on IgA+ cell number and immunoglobulin mRNA expression in the intestine of broilers

Abstract

Aflatoxin B₁ (AFB₁) is the most toxic group of mycotoxins produced by two species of the Aspergillus, common contaminants of food and animal feed. The purpose of our study was to determine the effect of AFB₁ on the number of IgA⁺ cell and immunoglobulin mRNA expression in the intestine of broilers. One hundred and fifty six one-day-old healthy Cobb broilers were randomly divided into the control group (the dosage of 0?mg/kg AFB₁) and AFB₁ group (the dosage of 0.6?mg/kg AFB₁) with three replicates per group and 26 birds per replicate for 21 days, respectively. After necropsy at 7, 14 and 21 days of age, duodenum, jejunum and ileum samples were taken for analyzing IgA⁺ cell by immunohistochemistry and IgA, pIgR, IgM and IgG mRNA expression by qRT-PCR. IgA⁺ cells were mainly distributed in the lamina propria of small intestinal mucosa in both groups at 14 and 21 days of age. A significant decrease in the number of IgA⁺ cells in the duodenum, jejunum and ileum was revealed in the AFB₁ group compared with that of the control group. The expression levels of IgA, pIgR, IgM and IgG mRNA in the intestinal mucosa were lower in the AFB₁ group than those in the control group at 14 and 21 days of age. Our data demonstrated that the dosage of 0.6?mg/kg AFB₁ in broiler diet reduced the number of IgA⁺ cell and the expression of IgA, pIgR, IgM and IgG mRNA in the small intestine.     

Ability of Lactobacillus plantarum MON03 to mitigate aflatoxins (B1 and M1) immunotoxicities in mice

Abstract

Aflatoxin B₁ (AFB₁) and M₁ (AFM₁) are mycotoxins produced by numerous Aspergillus species in pre- or post-harvest cereals and milk. AFB₁ and AFM₁ display a potent economic loss in livestock and also cause severe immunological problems. The aims of this study were to: evaluate a new AFB₁ and AFM₁-binding/degrading micro-organism for biological detoxification; examine its ability to degrade AFB₁ and AFM₁ in liquid medium; and evaluate its potential for in vivo preventative effects against AFB₁- and AFM₁-induced immunomodulation in mice. Lactobacillus plantarum MON03 (LP) isolated from Tunisian artisanal butter was found to display significant binding ability to AFB₁ and AFM₁ in PBS (i.e. 82% and 89%, respectively) within 24?h of incubation and able to tolerate gastric acidity, have strongly hydrophilic cells surface properties, and adhere efficacy to Caco-3 cells in vitro. The in vivo study was conducted using Balb/c mice that received by oral gavage vehicle (control), LP only (2?×?10⁹ CFU/L, ～2?g/kg BW), AFB₁ or AFM₁ alone (0.25 and 0.27?mg/kg, respectively), or AFB₁?+?LP or AFM₁?+?LP daily for 15 days. Compared to in control mice, treatments with AFB₁ and AFM₁ led to significantly decreased body weight gains, histopathological changes, and decrements in all hematologic and immune parameters assessed. Co-treatment with LP strongly reduced the adverse effects of each mycotoxin. In fact, the mice receiving AFB₁?+?LP or AFM₁?+?LP co-treatment displayed no significant differences in the assayed parameters as compared to the control mice. By itself, the bacteria alone had no adverse effects in the mice. From these data, it is concluded that the tested bacteria could be beneficial in biotechnology detoxification of contaminated food and feed for humans and animals.     

Immunoaffinity column/hplc determination of aflatoxin m1 in milk

Aflatoxin M₁ was selectively bound to a monoclonal antibody immobilized to an affinity column. After washing the column free of the milk matrix with 2 volumes of 10% MeOH in H₂O, the aflatoxin was eluted with 1 ml of MeOH. The eluate was subjected to high‐pressure liquid chromatography (HPLC) using a reverse‐phase column and fluorimetric detection. The detection limit was 50–100 ng l^‐1 of M₁ in milk. The method was tested with two different batches of affinity columns at a spiking level of 300 ng l^‐1 in milk, and showed recoveries of 98.7 and 79.3% and repeatabilities of 8.8 and 5.8% respectively. However, the method was found to be more reproducible when columns from the same batch were used (spiking levels 50 and 100 ng l^‐1). The detection limit could be further lowered to 10 ng l^‐1 by concentrating the eluate from the column. The results at low spiking levels ( ≤ 700 ng l^‐1) showed that although the recoveries were low, the repeatability was satisfactory enough to allow the implementation of the method with results corrected for recovery. The method compared well with chemical procedures and was fast, sensitive and more selective, with no interfering peaks appearing in the chromatograms.     

A novel signal amplification technology for ELISA based on catalyzed reporter deposition. Demonstration of its applicability for measuring aflatoxin B1

In an earlier communication we have described a novel signal amplification technology termed Super-CARD, which is able to significantly improve antigen detection sensitivity in conventional Dot-ELISA by approximately 10⁵-fold. The method utilizes hitherto unreported synthesized electron rich proteins containing multiple phenolic groups which, when immobilized over a solid phase as blocking agent, markedly increases the signal amplification capability of the existing CARD method (Bhattacharya, R., Bhattacharya, D., Dhar, T.K., 1999. A novel signal amplification technology based on catalyzed reporter deposition and its application in a Dot-ELISA with ultra high sensitivity. J. Immunol. Methods 227, 31.). In this paper we describe the utilization of this Super-CARD amplification technique in ELISA and its applicability for the rapid determination of aflatoxin B₁ (AFB₁) in infected seeds. Using this method under identical conditions, the increase in absorbance over the CARD method was approximately 400%. The limit of detection of AFB₁ by this method was 0.1 pg/well, the sensitivity enhancement being 5-fold over the optimized CARD ELISA. Furthermore, the total incubation time was reduced to 16 min compared to 50 min for the CARD method. Assay specificity was not adversely affected and the amount of AFB₁ measured in seed extracts correlated well with the values obtained by conventional ELISA.     

Detoxication of benzene and aflatoxin B1 with amino acid and peptide preparations in mice and chicken

Incubation of mouse and chicken splenocytes with amino acid or peptide preparationsin vitro increases cell resistance to benzene and aflatoxin B₁. Short-term (15 days) treatment of chicken with an amino acid mixture (aviamine) in combination with benzene also increased splenocyte resistance to toxinin vitro. By contrast, aviamine in combination with aflatoxin B₁ sharply decreased cell resistance to toxin. Glutamic acid possessed no such properties. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 10, pp. 419–421, October, 1999     

Genetic selection for aflatoxin B1 resistance influences chicken T-cell and thymocyte proliferation

Studies were conducted with two lines of chickens that were selected for high and low plasma protein concentrations in response to aflatoxin B₁ (AFB₁) exposure. The experiments were designed to determine genetic differences in the responses of T cells and thymocytes to the toxin. Chicks were orally administered AFB₁ at a rate of 0, 100, or 500 μg/kg body weight up to 21 days of age. At 4 weeks of age, concanavalin A (Con A, 2.5 μg/mL) stimulated T-cell proliferation was similar for untreated chicks from the low line (LL) and the high line (HL). However, AFB₁ reduced the responses of T cells with HL cells being more sensitive. In a second experiment, immature chickens were bled and peripheral blood lymphocytes were cultured with Con A and either 0, 3.125, 6.25, 12.5, or 25 μg/mL AFB₁. T cells from LL had greater responses to Con A than those from HL, and LL T-cells were also more resistant to in vitro AFB₁ exposure. Furthermore, thymocyte proliferation was greater for LL chicks; but when thymocytes were cultured with 25 μg/mL AFB₁, ³H-thymidine incorporation was similarly reduced in both lines. Cell cycle analysis indicated that there were more LL thymocytes in S phase, and the percentages for both lines decreased with AFB₁ treatment. Although there were no differences between the lines for percent G₂/M cells, AFB₁ treatment increased the percentages of thymocytes in G₂/M. These studies showed that selection for plasma protein response also changed T-cell and thymocyte proliferative activity.     

An improved indirect competitive Elisa for aflatoxin m1 in milk powders using novel monoclonal antibodies

Among three newly prepared monoclonal anti‐aflatoxin M₁ antibodies, named AM.1, AM.2 and AM.3, both AM.1 and AM.3 possessed high specificity and sensitivity towards aflatoxin M₁, while AM.2 exhibited lower specificity. Employing AM.3 and horseradish peroxidase‐labelled second antibody, an improved ELISA was developed with a detection limit of 1.0 pg ml^‐1 of aflatoxin M₁ in solution. The contents of aflatoxin M₁ in powdered milks suspended in water were assayed by the indirect ELISA. The detection limit was 5 pg g^‐1dry weight, and no clean‐up procedures were required. The reliability of the present ELISA with monoclonal antibody AM.3 was confirmed with reference powdered milks for aflatoxin M₁. A limited ELISA survey showed the presence of aflatoxin M₁ in commercial milk powders sampled in France, the US and Thailand at levels of 30–418 pg g^‐1, and it was confirmed by an improved HPLC analysis.     

Histamine-stimulated production of matrix metalloproteinase 1 by human rheumatoid synovial fibroblasts is mediated by histamine H1-receptors

The purpose of this study was to investigate the role of histamine in human rheumatoid synovial fibroblasts in the production of factors responsible for tissue remodelling and cartilage breakdown in rheumatoid arthritis. We examined the effects of histamine of tritiated thymidine incorporation, production of matrix metalloproteinase-1 (MMP-1), histamine H₁-receptor expression, phosphoinositide metabolism and intracellular calcium ion concentration ([Ca²⁺] _i ) in human rheumatoid synovial fibroblasts. Tritiated thymidine incorporation studies demonstrated that histamine markedly stimulated the proliferation of rheumatoid synovial fibroblasts. Immunofluorescence and Northern blot analyses revealed that proMMP-1 production was also stimulated by histamine. The levels of inositol phosphates and [Ca²⁺] _i in the cells were elevated in response to histamine, indicating that the cells expressed histamine H₁-receptors; and Northern blot analysis indicated that these H₁-receptors were up-regulated by histamine. In in situ hybridization, large amounts of histamine H₁-receptor mRNA were also detected in rheumatoid synovial tissue. These results suggest that the interaction between H₁-receptor expression in rheumatoid synovial fibroblasts and histamine secretion by mast cells and macrophages in the affected sites is an important event responsible for tissue remodelling and joint destruction in rheumatoid arthritis.     

Immortalization of human fibroblasts by liposome-mediated transfer of SV40 early region genes

Transfer of simian virus 40 (SV40) early region genes into normal cells is one of the most useful methods of obtaining immortalized cell lines. The SV40 early region encodes viral proteins that cause an increased, but finite, cellular proliferative potential; immortalization requires additional genetic changes. The following protocol describes plasmid-mediated transfer of SV40 early region genes into human fibroblasts and cell culture conditions designed to maximize the probability of obtaining an immortalized cell line. This approach may be used for any cell type.     

Immunochromatographic strip for ultrasensitive detection of fumonisin B1

A sensitive monoclonal antibody (mAb) (1H1) against fumonisin B₁ (FB₁) was produced in our laboratory. A rapid, simple, and an immunochromatographic strip has been developed for the ultrasensitive detection of FB₁ in corn samples. Under the optimized condition, the cut-off limits of test strips for FB₁ were found to be 50?ng/mL in 0.01?M PBS and 25?ng/mL in corn samples. Results could be obtained within 5 min. The results revealed that the developed method is a rapid, sensitive, and simple tool for the detection of FB₁.     

Increases of kinin B1 and B2 receptors binding sites after brain infusion of amyloid-beta 1-40 peptide in rats

Although numerous inflammation pathways have been implicated in Alzheimer's disease, the involvement of the kallikrein–kinin system is still under investigation. We anatomically localized and quantified the density of kinin B₁ and B₂ receptors binding sites in the rat brain after the infusion of amyloid-β (Aβ) peptide in the right lateral brain ventricle for 5 weeks. The conditioned avoidance test showed a significant reduction of memory consolidation in rats infused with Aβ (68.6 ± 20.9%, P < 0.05) when compared to control group (90.8 ± 4.1%; infused with vehicle). Autoradiographic studies performed in brain samples of both groups using [¹²⁵I]HPP-[des-Arg¹⁰]-Hoe-140 (150 pM, 90 min, 25 °C) showed a significant increase in density of B₁ receptor binding sites in the ventral hippocampal commissure (1.23 ± 0.07 fmol/mg), fimbria (1.31 ± 0.05 fmol/mg), CA1 and CA3 hippocampal areas (1.05 ± 0.03 and 1.24 ± 0.02 fmol/mg, respectively), habenular nuclei (1.30 ± 0.04 fmol/mg), optical tract (1.30 ± 0.05 fmol/mg) and internal capsule (1.26 ± 0.05 fmol/mg) in Aβ group. For B₂ receptors ([¹²⁵I]HPP-Hoe-140, 200 pM, 90 min, 25 °C), a significant increase in density of binding sites was observed in optical tract (2.04 ± 0.08 fmol/mg), basal nucleus of Meynert (1.84 ± 0.18 fmol/mg), lateral septal nucleus – dorsal and intermediary portions (1.66 ± 0.29 fmol/mg), internal capsule (1.74 ± 0.19 fmol/mg) and habenular nuclei (1.68 ± 0.11 fmol/mg). In control group, none of these nuclei showed [¹²⁵I]HPP-Hoe-140 labeling. This significant increase in densities of kinin B₁ and B₂ receptors in animals submitted to Aβ infusion was observed mainly in brain regions related to cognitive behavior, suggesting the involvement of the kallikrein–kinin system in Alzheimer's disease in vivo.     

Germline copy number variation of genes involved in chromatin remodelling in families suggestive of Li-Fraumeni syndrome with brain tumours

Germline alterations of the tumour suppressor TP53 gene are detected approximately in 25% of the families suggestive of Li-Fraumeni syndrome (LFS), characterised by a genetic predisposition to a wide tumour spectrum, including soft-tissue sarcomas, osteosarcomas, premenopausal breast cancers, brain tumours, adrenocortical tumours, plexus choroid tumours, leukaemia and lung cancer. The aim of this study was to determine the contribution of germline copy number variations (CNVs) to LFS in families without detectable TP53 mutation. Using a custom-designed high-resolution array CGH, we evaluated the presence of rare germline CNVs in 64 patients fulfilling the Chompret criteria for LFS, but without any detectable TP53 alteration. In 15 unrelated patients, we detected 20 new CNVs absent in 600 controls. Remarkably, in four patients who had developed each brain tumour, the detected CNV overlap the KDM1A, MTA3, TRRAP or SIRT3 genes encoding p53 partners involved in histone methylation or acetylation. Focused analysis of SIRT3 showed that the CNV encompassing SIRT3 leads to SIRT3 overexpression, and that in vitro SIRT3 overexpression prevents apoptosis, increases G2/M and results in a hypermethylation of numerous genes. This study supports the causal role of germline alterations of genes involved in chromatin remodelling in genetic predisposition to cancer and, in particular, to brain tumours.     

Effect of oral supplementation of Lactobacillus reuteri in reduction of intestinal absorption of aflatoxin B(1) in rats

The goals of this work were to assess the ability of Lactobacillus reuteri to bind aflatoxin B(1) in the intestinal tract and determine its effect on intestinal absorption of the toxin dispensed in either single or multiple doses in a murine model. Male Wistar rats were used, and two experiments were conducted after bacteria were implanted. Experiment one involved a single-oral dose of toxin, and the subsequent flow cytometric analysis of bacteria isolated from the small intestine and treated with specific FITC-labeled AFB(1) antibodies. The second experiment was carried out supplying the toxin in 7 oral sub-doses, and the later quantification of AFB(1)-Lys adducts in blood samples by ELISA assay. The results demonstrated that L. reuteri was able to bind AFB(1) in the intestinal tract, mostly in the duodenum. Furthermore, the AFB(1)-Lys adducts were present at significantly lower levels in those animals receiving AFB(1) plus bacteria than in those receiving only AFB(1). Our findings confirm that probiotic bacteria could act as biological barriers in normal intestinal conditions thereby reducing the bioavailability of AFB(1) ingested orally in a single or multiple doses, thus avoiding its toxic effects.