Ladislav Fukal

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.

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.

A survey of cereals, cereal products, feedstuffs and porcine kidneys for ochratoxin A by radioimmunoassay.

A commercial kit for the radioimmunochemical determination of ochratoxin A has been validated for application to various foods and feedstuffs. Using this kit a survey was carried out for the occurrence of ochratoxin A in Czechoslovak cereals, cereal products, feedstuffs and pig kidneys. The results show that the most contaminated are feedstuffs and cereals, in 26.7% and 8.8% of the samples of which, respectively, the ochratoxin A level exceeded 20 micrograms/kg. None of the cereal products and 4.2% of the pig kidneys unfit for human consumption by visual examination possessed ochratoxin A concentration higher than 5 micrograms/kg. Of all the samples analysed, 57% contained no detectable toxin (less than 1 microgram/kg).

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.

Development of an ELISA specific for Listeria monocytogenes using a polyclonal antibody raised against a cell extract containing internalin B

We have developed a new enzyme-linked immunosorbent assay (ELISA) that is specific to the foodborne pathogenic micro-organism Listeria monocytogenes. It is based on an antibody raised against an L. monocytogenes cell preparation optimized for extraction of internalin B. Only in a sandwich ELISA format was the protein A-purified antibody specific to L. monocytogenes. In a competitive ELISA format, the antibody recognizes other Listeria species. The sandwich ELISA shows no recognition of L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, or L. grayii. It has a minimum detectable level for L. monocytogenes of log10 6.37 cfu ml−1 in pure culture, is reproducible, and is unaffected by the presence of high numbers (approximately log10 8.0 cfu ml−1) of the other Listeria species. Possible reasons for the format-dependent specificity are discussed. When the ELISA was applied to milk samples inoculated with L. monocytogenes reference material (5 cfu ml−1), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of food samples.

Selection of the separation step in the radioimmunoassay for aflatoxin B1 using125I as a marker

Aflatoxin B1 was assayed by radioimmunoassay (RIA) using125I-labelled antigen. The immunoreactivity of the radioligand applied is very close to the immunoreactivity of free aflatoxin B1. The logit-log analysis was used to select the best separation of free and bound radioligand. The adsorption of the free radioligand on dextran-coated charcoal was found to be superior from the viewpoint of the assay sensitivity and accuracy. The detection limit of aflatoxin B1 was about 10 pg per tube. The assay accuracy was estimated to 3.3% in intraassay and to 7.0% in interassay.

Radioimmunoassay of aflatoxin M1

Radioimmunoassay for the differential determination of aflatoxin M1 has been developed. It is based on the use of antiserum having almost the same affinity to both aflatoxins B1 and M1 /Ka for aflatoxin B1=2.0×109 and Ka for aflatoxin M1=2.1×109/ and making possible their simultaneous determination. Aflatoxin B1 content is determined specifically by a commercial RIA, and aflatoxin M1 concentration is calculated as the differences between these two assays.

Choice of procedure conditions for radioimmunoassay of aflatoxin B1 using125I as a marker and dextrancoated charcoal as a separation matrix

Assay conditions for the radioimmunoassay for aflatoxin B1 using125I-radiolabel and dextran-coated charcoal for the separation of free and bound radioligand were optimized. Casein was chosen as the best protecting protein /in contrast with human serum albumin, γ-globulin and gelatine/. The most suitable incubation conditions are at 4°C for 18 h in darkness, radioligand sorption on the dextrancoated charcoal takes place 30 min at 4°C and the antiserum is diluted in order to reach zero specific binding in the range between 35 and 50%.

Proteolytic activity and immunoreactivity of chemically modified Papain

ZusammenfassungEs wurde der Einfluß verschiedener chemischer Modifizierung des Papains auf dessen proteolytische Aktivität und Immunreaktivität untersucht. Die Modifikation des Papains mit Dextran T 2000 1äßt die proteolytische Aktivität bei steigender Dextran-oxidation und mit der Menge des gebundenen Dextrans abnehmen, wobei die Immunreaktivität, nephelometrisch bestimmt, unverändert bleibt. Bei der Modifizierung von Papain mit Glutaraldehyd und Formaldehyd werden beide Aktivitäten, auch bei sehr niedrigen Konzentrationen der Reagentien, stark erniedrigt. Das acetylierte Papain zeigt ein scharfes Maximum der proteolytischen und der immunchemischen Aktivität bei demselben Grad der Enzymacetylierung. Die Modifizierung von Papain mit der Diazobenzosulfonsäure verursacht einen hohen Anstieg der Immunreaktivität und einen nur geringen der proteolytischen Aktivität.SummaryThe effect of different chemical modifications of papain on the proteolytic activity and immunoreactivity has been studied. Modification with Dextran T 2000 caused increasing decline in proteolytic activity both with increasing degree of dextran oxidation and amount of bound dextran, whilst the immunoreactivity determined by nephelometry remained unchanged. Modification of papain with glutaraldehyde and formaldehyde causes rapid drops in both activities, even at very low concentrations of agents. Acetylation of papain showed expressive maxima of both proteolytic and immunochemical activities at the same degree of enzyme acetylation. Modification with diazobenzensulphonic acid caused a high increase in immunoreactivity and a small increase in proteolytic activity.