Heterologous screening of hybridomas for the development of broad-specific monoclonal antibodies against deoxynivalenol and its analogues

Hapten heterology was introduced into the steps of hybridoma selection for the development of monoclonal antibodies (MAbs) against deoxynivalenol (DON). Firstly, a novel heterologous DON hapten was synthesised and covalently coupled to proteins (i.e. bovine serum albumin (BSA), ovalbumin and horseradish peroxidase) using the linkage of cyanuric chloride (CC). After immunisation, antisera from different DON immunogens were checked for the presence of useful antibodies. Next, both homologous and heterologous enzyme-linked immunosorbent assays were conducted to screen for hybridomas. It was found that heterologous screening could significantly reduce the proportion of false positives and appeared to be an efficient approach for selecting hybridomas of interest. This strategy resulted in two kinds of broad-selective MAbs against DON and its analogues. They were quite distinct from other reported DON-antibodies in their cross-reactivity profiles. A unique MAb 13H1 derived from DON-CC-BSA immunogen could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyl-DON > DON > nivalenol, with IC50 ranging from 1.14 to 7.69 mu g/ml. Another preferable MAb 10H10 generated from DON-BSA immunogen manifested relatively similar affinity to DON, 3-acetyl-DON and 15-acetyl-DON, with IC50 values of 22, 15 and 34 ng/ml, respectively. This is the first broad-specific MAb against DON and its two acetylated forms and thus it can be used for simultaneous detection of the three mycotoxins

Capacitive sensor based on molecularly imprinted polymers for detection of the insecticide imidacloprid in water

This manuscript reports on the development of a capacitive sensor for the detection of imidacloprid (IMD) in water samples based on molecularly imprinted polymers (MIPs). MIPs used as recognition elements were synthesized via a photo-initiated emulsion polymerization. The particles were carefully washed using a methanol (MeOH) / acetic acid mixture to ensure complete template removal and were then dried. The average size of the obtained particles was less than 1 mu m. The imprinting factor (IF) for IMD was 6 and the selectivity factor (alpha) for acetamiprid, clothianidin, thiacloprid and thiamethoxam were 14.8, 6.8, 7.1 and 8.2, respectively. The particles were immobilized on the surface of a gold electrode by electropolymerization. The immobilized electrode could be spontaneously regenerated using a mixture of MeOH/10 mM of phosphate buffer (pH = 7.2)/triethylamine before each measurement and could be reused for 32 times. This is the first-time that automated regeneration was introduced as part of a sensing platform for IMD detection. The developed sensor was validated by the analysis of artificially spiked water samples. Under the optimal conditions, the linearity was in the range of 5-100 mu M, with a limit of detection (LOD) of 4.61 mu M

Corrigendum to ‘Dietary aflatoxin B1 (AFB1) reduces growth performance, impacting growth axis, metabolism, and tissue integrity in juvenile gilthead sea bream (Sparus aurata)’. Aquaculture, volume 533, 25 February 2021, 736189

The authors regret the errors in a few table references within the text. Specifically, it should reads as follows within the following subsections/ page: 3.2. Blood analysis (page 5)info:eu-repo/semantics/publishedVersio

Gel electrophoresis separation and origins of light emission in fluorophores prepared from citric acid and ethylenediamine

We investigated light emission of hydrothermally treated citric acid and ethylenediamine (EDA) with various precursor ratios using gel-electrophoresis. We show that this relatively simple approach can deliver significant insights into the origins of photoluminescence. We found that products of the synthesis consist of both positively and negatively charged species and exhibit large dispersion in electrophoretic mobility (i.e. charge-to-size ratio). We observed that despite the large dispersion of the reaction products the blue light emission is confined to discrete bands clearly identifiable in the gel. We demonstrate clear evidence that this emission originates from the negatively charged light molecular fraction with the highest mobility which shows no excitation-dependent light emission. This molecular fluorophore exhibits spectral characteristics similar to previously reported 1,2,3,5-tetrahydro-5-oxo-imidazo[1,2-a]pyridine-7-carboxylic acid (IPCA). Secondary gel electrophoresis run performed on the bands extracted from the first run indicates that no further separation takes place. On the basis of our experimental results, we conclude that relatively stable binding exists between IPCA and EDA-derived product. Thus, the products of the reaction contain IPCA both in molecular form and in complexes with EDA-derived products. We conclude that excitation-dependent emission is related to the fluorophore binding to the positively charged EDA-derived products with a positive charge

Evaluation of mycotoxin content in soybean (Glycine max l.) grown in Rwanda

Soybean is a critical food and nutritional security crop in Rwanda. Promoted by the Rwandan National Agricultural Research System for both adults and as an infant weaning food, soybean is grown by approximately 40% of households. Soybean may be susceptible to the growth of mycotoxin-producing moulds; however, data has been contradictory. Mycotoxin contamination is a food and feed safety issue for grains and other field crops. This study aimed to determine the extent of mycotoxin contamination in soybean, and to assess people’s awareness on mycotoxins. A farm-level survey was conducted in 2015 within three agro-ecological zones of Rwanda suitable for soybean production. Soybean samples were collected from farmers (n=300) who also completed questionnaires about pre-and post-harvest farm practices, and aflatoxin awareness. The concentration of total aflatoxin in individual soybean samples was tested by enzymelinked immunosorbent assay (ELISA) using a commercially-available kit. Other mycotoxins were analyzed using liquid chromatography-mass spectrometry (LCMS/ MS) on 10 selected sub samples. Only 7.3% of the respondents were aware of aflatoxin contamination in foods, but farmers observed good postharvest practices including harvesting the crop when the pods were dry. Using enzyme-linked immunosorbent assay (ELISA), only one sample had a concentration (11 μg/kg) above the most stringent EU maximum permitted limit of 4 μg/kg. Multi-mycotoxins liquid chromatography-mass spectrometry (LC-MS/MS) results confirmed that soybeans had low or undetectable contamination; only one sample contained 13μg/kg of sterigmatocystine. The soybean samples from Rwanda obtained acceptably low mycotoxin levels. Taken together with other studies that showed that soybean is less contaminated by mycotoxins, these results demonstrate that soybean can be promoted as a nutritious and safe food. However, there is a general need for educating farmers on mycotoxin contamination in food and feed to ensure better standards are adhered to safeguard the health of the consumers regarding these fungal secondary metabolites.Key words: soybean, safety, mould, aflatoxin, mycotoxins, sterigmatocystine, ELISA, LC-MS/MS, Rwand

Deoxynivalenol content in wheat dust versus wheat grain: a comparative study

The present study, set up in the growing season 2011-2012, was designed to obtain quantitative data on the occurrence of deoxynivalenol in wheat grain and the corresponding wheat dust. The field experiment consisted of a complete randomised block design with five wheat varieties sown on a field on which maize was grown in the previous season. The impact of the tillage method and the influence of the wheat variety resistance on the deoxynivalenol content of wheat and wheat dust were investigated. The accumulation of deoxynivalenol in wheat dust was confirmed and a sigmoidal relationship between the deoxynivalenol content in wheat dust versus wheat grain was determined. Deoxynivalenol reduction was obtained by ploughing and by sowing moderately resistant wheat varieties. As wheat dust provides equal results and solves the problem of heterogeneity during sampling of conventional wheat matrix, the sampling of wheat dust can be considered as a promising alternative

Immunomagnetic microbeads for screening with flow cytometry and identification with nano-liquid chromatography mass spectrometry of ochratoxins in wheat and cereal

Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography–quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants