Igor Hochel

Deoxynivalenol and its conjugates in beer: A critical assessment of data obtained by enzyme-linked immunosorbent assay and liquid chromatography coupled to tandem mass spectrometry

Enzyme-linked immunosorbent assays (ELISAs) are often employed for the control of deoxynivalenol (DON) in barley and other intermediates involved in beer production chain. Because of the occurrence of high levels of DON-3-glucoside (DON-3-Glc) in malt and beer that have been reported for the first time in our earlier study, research focused on the accuracy of DON determination by immunoassays in cereal-based matrices has been initiated. DON-3-Glc was strongly cross-reacting in all examined commercial ELISA test kits (Ridascreen) DON (R-Biopharm), Veratox 5/5 DON) (Neogen Corporation), Deoxynivalenol EIA (Euro-Diagnostica), and AgraQuant) DON Assay 0.25/5.0 Test Kit (Romer Labs). The highest overestimation in beer analysis, up to 1000%, when taking the DON content determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as a reference method, was obtained by AgraQuant assay. Besides of DON-3-Glc and 3- and 15-acetyldeoxynivalenol (ADONs), also other, not known yet, matrix components contributed to false positive results. Similar phenomenon, although in a lesser extent due to lower content of these substances, was observed for using ELISA in the analysis of wheat. The relationship between a way of sample handling and DON overestimation was demonstrated; higher ELISA response was measured in an aqueous extract compared to that prepared by acetonitrile-water (84:16, v/v). Most of cross-reacting co-extracts were removed by MycoSep# 226 cartridge, what leads us to the hypothesis on the presence of currently unknown cross-reactive species.

Immunochemical determination of gluten in malts and beers

The gluten content in different varieties of barley and malts, and in different types of beers, was determined by a ‘sandwich’ enzyme immunoassay (RIDASCREEN® Gliadin kit). The gluten levels in barley wheat, rye and spelt malts ranged 18.8–45.0, 44.0–68.0, 41.6 and 21.2 g kg–1, respectively. When various types of beer were compared, the gluten concentration increased as follows: alcohol-free beer (<3.0), lager beers (<3.0–8.7 mg l–1), stouts (9.0–15.2 mg l–1) and wheat beers (10.6–41.2 mg l–1). When 10 Czech lager beers were analysed, using both sandwich and competitive ELISA, the results showed that the latter method provided values several times higher than the former. Gluten balance was carried out during the brewing process, starting from the raw materials and terminating at the final beer. Gluten levels decreased due to precipitation during the mashing process, primary and secondary fermentation and, lastly, as a result of adsorption during beer stabilization. The gluten content in beer is, thus, approximately three orders of magnitude lower than in the raw malt.

Occurrence of Cronobacter spp. in retail foods

Aims:  To study the occurrence of Cronobacter spp. in foods and to investigate the phenotypic properties of the strains isolated.

Detection of Campylobacter species in foods by indirect competitive ELISA using hen and rabbit antibodies

Abstract The competitive enzyme immunoassays for detection of Campylobacter jejuni, C. coli and C. fetus subsp. fetus have been developed. Rabbit and hen immunoglobulins were prepared for these purposes. The working conditions of ELISAs, such as the concentrations of immunoreactants, incubation temperatures and time, and the composition of the substrate have been established. The detection limits were in the range 5.0 104–3.2 106 cfu/ml. The application of chemiluminescent substrates did not result in any significant improvement of the assays detectability and sensitivity. Prepared antibodies showed rather high specificity and cross-reactivity profiles, and both rabbit and hen immunoglobulins were similar. Only IgY to C. jejuni cross-reacted with seven strains of C. jejuni and two other Campylobacter spp. A limited number of naturally and artificially contaminated food samples were tested. The results obtained by means of an enzyme immunoassay were compared with those obtained from PCR or commercially available Singlepath® Campylobacter GLISA-Rapid Test. Poultry products were naturally contaminated with Campylobacters. The wild species were identified as C. jejuni and C. coli.

Development of an Indirect Competitive ELISA for Detection of Campylobacter jejuni subsp. jejuni O:23 in Foods

An indirect enzyme immunoassay for rapid detection ofCampylobacter jejuni subsp.jejuni O:23 has been developed. Optimum concentrations of immobilized cells, polyclonal chicken IgY, and rabbit anti-IgY antibody-horseradish peroxidase conjugate were 3.1 CFU/nL, 10 µg/mL, and 8 µg/mL, respectively. Under such conditions, the detection limit reached 50 CFU/µL, limit of quantification being 480 CFU/µL. By testing 5 chromogens.viz. 1,2-benzenediamine, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3′,5,5′-tetramethylbenzidine, bi(4,4′-anisidine) and 3-methyl-2-benzothiazolinone hydrazone, in horseradish peroxidase substrate, 1,2-benzenediamine or 3,3′,5,5′-tetramethylbenzidine as H-donors in the enzyme substrate provided the highest ELISA sensitivity. The applied polyclonal antibody was specific for homogeneous antigen. The cross-reactions were observed only with one strain ofC. sputorum subsp.sputorum (21.5 %) and with G+ bacteriumMicrococcus luteus (6.1 %). Preliminary tests have been performed with a limited number of artificially contaminated food samples. No matrix effects on the ELISA sensitivity were observed. The results (by means of ELISA) were comparable with those given by both a standard cultivation method performed according toČSN ISO 10272 and commercially available Singlepath® Campylobacter GLISA-Rapid Test.

Enzyme immunoassay of histamine in foods

A competitive enzyme immunoassay of histamine in foodstuffs has been developed using a monoclonal antibody against a histamine‐benzoquinone adduct. In this assay, histamine present in food samples was treated with 1,4‐benzoquinone to form histamine‐benzoquinone by a simple and quick reaction and a histamine‐benzoquinone‐horse‐radish peroxidase conjugate was used as the labelled hapten. The apparent association constant (Ka) of the antibody used was 3.6 ×106 l/mol and Gibbs’ energy of the immune complex formation has been estimated to find the optimal incubation time of the assay. The method enabled determination of histamine in fish, cheese, wine and beer at a concentration as low as 7 ng/ml with an accuracy of ± 15%. The recovery of the immunoassay was 88.9–114%. Cross‐reactivities of histidine, tyramine, tryptamine and its derivatives were lower than 0.001% and did not affect the assay.

Differentiation of Cronobacter spp. by tryptic digestion of the cell suspension followed by MALDI-TOF MS analysis

Intact cell MALDI-TOF mass spectrometry is a rapid tool for the identification and classification of microorganisms, now widely used even in clinical laboratories. However, its distinctive power is not sufficient for some closely-related species. The genus Cronobacter, formerly known as Enterobacter sakazakii, contains such species. In this work, a new method for the differentiation of five Cronobacter species is presented involving the tryptic digestion of cytoplasmatic proteins followed by MALDI mass spectrometry analysis. A database was developed for use in Bruker Biotyper software including 52 reference spectra and tested on a set of 45 samples with an overall accuracy of about 80%. The possibility of measurement automation and the short time and low cost requirements of this method compared to those of biochemical tests or PCR methods make it a supplementary option to intact cell MALDI, providing additional information about the differentiation of problematic species.

Application of Mouse Antibodies to Somatic Antigen for Detection of Salmonella enteritidis by Competitive ELISA

Competitive ELISA estimation based on application of polyclonal mouse antibodies to somatic antigen O:9, 12 was developed. The optimization of the protocol is reported. The optimal concentration of immobilized somatic antigen O:9, 12 was found to be 4.9 2 104 cells ml−1; optimal concentration of mouse IgG was 6.25 w g ml−1; and the optimal concentration of peroxidase labelled antibody to mouse IgG was 8 w g ml−1. The tested antibody exhibited neither cross reactions with chosen strains (serotypes) of salmonellas group 04 (B), 07 (C1), 08 (C2-C3), nor with members of Enterobacteriaceae: Escherichia coli, Klebsiella pneumonia, Citrobacter freundii and non-fermenting bacterium Pseudomonas fluorescens. Application of chemiluminiscent substrates increased the sensitivity of S. enteritidis detection up to three times. Competitive ELISA tested on model samples produced results comparable with standard cultivation techniques for Salmonella spp.

Determination of DDT in model samples by using solid-phase extraction and indirect competitive enzyme immunoassay

Two-step indirect competitive enzyme immunoassay of DDT has been developed. The limit of detection and limit of quantification were 0.3 nmol l−1 and 4.2 nmol l−1, respectively. To shorten the analysis time, the one-step enzyme immunoassay has been developed by replacing anti-DDT antibody with the complex anti-DDT antibody-HRP. The anti-DDT antibody-HRP conjugate was prepared by periodate method. A concentration of the preparation was 1.9 mg·ml−1 and molar ratio Px/IgG was 1.87. The detection limit of the one-step ELISA reached 0.3 nmol l−1, but the sensitivity of assay was very poor. The two-step enzyme immunoassay of DDT has been adapted for the chemiluminescent detection of the complex antigen-antibody. The detectability of chemiluminescent ELISA was comparable with that of chromogenic ELISA. A limited number of the artificially contamined samples have been tested. All samples were prepared by solid-phase extraction by using the commercial Agilent Zorbax SPE C18 columns. A background was observed in most tested samples. The recoveries obtained from the analysis of DDT spikes reached values between 89.0–161.0%, and 81.5–111.6% at standard addition 50 μg·kg−1, and 200 μg·kg−1, respectively. The overestimated amounts of DDT were determined in spinach and nectarines at standard addition 50 μg·kg−1.