Pavel Rauch

Strip-based immunoassay for rapid detection of thiabendazole.

There is increased interest in the investigation and implementation of rapid screening methods for detection of pesticide residues. This study reports development of an immunostrip test for thiabendazole detection based on indirect competitive principle using carbon particles as a label. Nitrocellulose membrane strip was coated with a thiabendazole-protein conjugate in the defined test zone. In flow of an antibody-carbon complex and thiabendazole along the strip, the intensity of black colour formed in the test line reflected the thiabendazole concentration and semi-quantitative estimation could be carried out visually. The optimized test was accomplished within 10 min and the visual detection limit was achieved 0.25 ng mL(-1) of standard sample. Moreover, immunostrip was evaluated quantitatively using scanning densitometry. Based on standard curve, the detection limit of the proposed test was as low as 0.08+/-0.03 ng mL(-1) with an IC(50) value of 0.60+/-0.08 ng mL(-1) and a linear working range of 0.11-4.13 ng mL(-1). Results of testing precision, stability, and specificity demonstrated that the assay provided a reliable performance. This immunostrip was applied to analysis of spiked fruit juices in range of 0.05-5 mg L(-1). Matrix interferences were avoided by simple dilution of samples. Both visual and instrumental evaluations indicated a good agreement with results obtained by ELISA. Recoveries from juices were from 81.9 to 123.6% and relative standard deviations ranged from 9.9 to 19.3%. The developed strip offers potential as a useful rapid and simple method for screening of thiabendazole in fruit juices at levels far below the maximum residue limits.

Immunochromatographic colloidal carbon-based assay for detection of methiocarb in surface water.

A simple and rapid immunochromatographic assay for a sensitive and inexpensive monitoring of methiocarb in surface water was developed using a binding inhibition format on a membrane strip. In the assay, detection reagent consisted of anti-methiocarb antibody and colloidal carbon-labelled secondary antibody. Methiocarb-ovalbumin conjugate was immobilized in a test line of the strip as a capture reagent. Colour intensity of the test line in methiocarb-positive assay was visually distinguishable from that of negative sample within 10min. The optimized semi-quantitative method provided a visual detection limit of 0.5ngmL(-1). Cross-reactions with other carbamate pesticides were not found (<1%). Only a negligible matrix effect of surface water was recognized. In parallel analyses of spiked water samples, the assay results were in a good agreement with those of ELISA. The stability test indicated the strips could be used at least 2 months without change in performance. All characteristics of the visually evaluated assay mentioned above were verified by instrumental quantification of colour intensity in test lines. The developed immunochromatographic assay offers potential as a useful on-site screening tool for environmental analysis.

Chemiluminescent flow sensor for the determination of Paraoxon and Aldicarb pesticides

Abstract A chemiluminescence-based flow method for the determination of some organophorus and carbamate pesticides based on inhibition of acetylcholinesterase was developed. Acetylcholinesterase in solution or immobilized on methacrylate beads (Eupergit C) was coupled to choline oxidase and peroxidase immobilized on Eupergit C. In this system choline formed by acetylcholinesterase was oxidized by choline oxidase and the H 2 O 2 produced was determined via the luminol/peroxidase luminescent reaction. The detection limits (3 σ) for Paraoxon and Aldicarb were 0.75 μg 1 −1 and 4 μg 1 −1 , respectively, when soluble acetylcholinesterase was used under the following optimized experimental conditions: 56 μM luminol in working solution, sample volume 60 μl, flow-rate 0.3 ml min −1 and 60 min incubation time. The flow sensor device using all the three enzymes in the immobilized form had a higher detection limit of 125 μg 1 −1 for Paraoxon. The mid-range relative standard deviation ( n =10) using 1 mM standard substrate solution was 3.7%. The recovery from contaminated samples (soil, vegetables) varied from 81 to 108%. The results obtained by the developed methods were in good agreement with those obtained by a commonly used colorimetric test.

Determination of PAHs in various smoked meat products and different samples by enzyme immunoassay

An enzyme immunoassay was used to determine benzo[a ]pyrene (BaP) in smoked meat products and other samples of food and environmental origin. The method used has a detection limit (3 σ) of 0.1 μg kg−1 and a coefficient of variation less than 10%. The main aim of the study was to compare the possible influence of different smoking processes and packaging material on the amount of BaP deposited on smoked meat product, mainly different sausages. The lowest amount of BaP was found when smoke produced by steam in the indirect method smoking-chamber was used. A slightly protective effect of polyamide casing was noted. © 1999 Society of Chemical Industry

Immunoprobes for thermally-induced alterations in whey protein structure and their application to the analysis of thermally-treated milks

Polyclonal antisera have been raised to the whey proteins α-lactalbumin [α-La] and β-lactoglobulin [β-Lg], variants A and B. These antibody preparations have been used to develop enzyme-linked immunosorbent assays (ELISAs) for each of these proteins, which had limits of detection of 13 ng/ml [α-La], 27 ng/ml [β-Lg, variant A], and 20 ng/ml [β-Lg, variant B]. The α-La ELISA did not show any cross-reaction with β-Lg, and neither of the β-Lg ELISAs showed a cross-reactivity with α-La. However, despite the almost identical sequences of variants A and B of β-Lg, the variant A ELISA had a cross-reactivity of 66% with variant B, whilst the variant B ELISA had a cross-reactivity of more than 200% with variant A. The effect of thermal treatment on the immunoreactivity of purified whey proteins was studied by ELISA and related to changes in secondary and tertiary structure determined using CD and fluorescence spectroscopy. The immunoreactivity of α-La determined by ELISA decreased on heating above 90°C, these changes coinciding with the protein denaturation as indicated by a loss of secondary structure. In contrast, the ELISA immunoreactivity of both β-Lg variants increased after heating, a change that also coincided with changes in β-Lg secondary and tertiary structure as determined by intrinsic fluorescence of the protein. Similar thermally-induced changes in whey protein immunoreactivity were observed following heat-treatment of raw milk, the immunoreactivity of α-La being reduced whilst that of the β-Lg variants increased. When used in combination these ELISAs were able to discriminate between milks which had been pasteurized or subjected to more severe heat-treatments such as sterilization and ultra heat treatments (UHT). These data demonstrate that such immunoassays have the potential to be used as quality control methods for determining the thermal history of milks.

Combined Immunomagnetic Separation and Detection of Salmonella enteritidis in Food Samples

A model system for immunochemical detection of Salmonella enteritidis has been developed, using immunomagnetic separation (IMS) with both commercially‐available and laboratory‐prepared antibodies, followed by an enrichment stage and end‐point detection by ELISA. IMS alone gave an average of 77% recovery of cells artificially inoculated into the food, and a range of 20–110% recovery, depending on food type. The combined model system (IMS‐ELISA) enables detection of either 10 cells ml−1 (3 h enrichment after IMS). Cross‐reactivity of antibodies with Citrobacter was decreased by using two different immunochemical steps: IMS with monoclonal antibody‐coated beads, and a ‘sandwich’ ELISA with polyclonal capture antibody and monoclonal detector antibody. Discussion is presented on the best food preparation method, optimal substrate type for the ELISA and on the potential of IMS.

The potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of biogroups of Cronobacter sakazakii

RATIONALE The bacterial genus Cronobacter was established quite recently, in 2008. Therefore, its systematic classification is still in progress as well as the risk assessment of Cronobacter strains. The possibility of rapid identification within the biogroup level has an essential epidemiological significance. We examined the potential of mass spectrometry to accomplish this task on species Cronobacter sakazakii comprising eight different biogroups. METHODS Members of all Cronobacter sakazakii biogroups were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using intact cells. Analyses were performed on a Biflex IV MALDI-TOF mass spectrometer in the range of 2000 to 20 000 Da in linear mode with an accelerated voltage of 19 kV. RESULTS Optimal conditions for a proper identification of biogroups, such as suitable cultivation media or growth time of bacteria, were investigated. The biomarker patterns characterizing each of the Cronobacter sakazakii biogroups were obtained. The established identification protocol was applied to ten previously non-identified strains and their biogroups were successfully determined. CONCLUSIONS The presented work is the first report of successful and rapid bacterial biogroup taxonomy classification using MALDI-TOF-MS that could substitute demanding biochemical testing.

Luminescent enzymatic flow sensor for d- and l-lactate assay in beer

Abstract A bioluminescent flow sensor, previously developed for the determination of both d- and l-lactate in clinical samples, was utilized to carry out the same assay in beer. The sensor monitored the reduced form of nicotinamide adenine dinucleotide, produced by nylon-immobilized d- and l-lactate dehydrogenase, by means of bacterial bioluminescent enzymes immobilized on a separate nylon coil. The preparation of beer samples was very simple as only a modification of pH and a dilution were necessary. The recoveries ranged from 91% to 104%, and the relative standard deviations at the 1 mmol 1–1 level were 4.6% and 6.7% for l- and d-lactate respectively. The response was linear in the range 0.1–10 mmol 1–1 for both d- and l-lactate. The total amount of lactate determined by bioluminescent biosensor (x) and by HPLC (y) showed a very good correlation (y=0.654 x+88.1, n=29, r=0.918). The flow injection system developed allowed the determination of not only the total but also the individual contents of d- and l-lactate in beer, and the timely discovery of the unwanted presence of lactic acid bacteria.

Development of a radioimmunoassay for papain

Various well-tried radioimmunoassay (RIA) techniques were compared for the quantitation of papain. The evaluation of individual assays was performed by logit-log analysis. The most compatible analytical steps were combined in order to obtain the optimal analytical conditions of the assay. The preferred RIA involves papain labelling with lactoperoxidase, a double antibody method as the separation step and a 24 h incubation period at 2 degrees C. It permits the detection of 16 ng of papain per tube. In contrast, a method using immobilized antibody was satisfactory for rapid quantitation of papain with quite acceptable accuracy.

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.