Sergei A. Eremin

Immunochemical methods for rapid mycotoxin detection: evolution from single to multiple analyte screening: a review.

This review focuses on recent developments in immunochemical methods for detection of mycotoxins, with a particular emphasis on simultaneous multiple analyte determination. This includes high-throughput instrumental analysis for the laboratory environment (microtitre plate enzyme-linked immunoabsorbant assay (ELISA), different kinds of immunosensors, fluorescence polarization immunoassay, and capillary electrophoretic immunoassay), as well as rapid visual tests for on-site testing (lateral-flow, dipstick, flow-through and column tests). For each type of immunoassay, perspectives for multiple analyte application are discussed and examples cited.

A simple, rapid and high-throughput fluorescence polarization immunoassay for simultaneous detection of organophosphorus pesticides in vegetable and environmental water samples

A simple, rapid and high-throughput fluorescent polarization immunoassay (FPIA) for simultaneous determination of organophosphorus pesticides (OPs) using a broad-specificity monoclonal antibody was developed. The effects of tracer structure, tracer concentration, antibody dilution, methanol content and matrix effect on FPIA performance were studied. The FPIA can detect 5 OPs simultaneously with a limit of detection below 10 ng mL(-1). The time required for the equilibrium of antibody-antigen interaction was less than 10 min. The recovery from spiked vegetable and environmental samples ranged from 71.3% to 126.8%, with the coefficient of variations ranging from 3.5% to 14.5%. The developed FPIA was applied to samples, followed by confirmation with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. The developed FPIA demonstrated good accuracy and reproducibility, and is suitable for rapid and high-throughput screening for OP contamination with high-efficiency and low cost.

Enzyme immunoassays for s-triazine herbicides and their application in environmental and food analysis

Abstract Seventeen rabbits and forty mice were immunized with immunogens prepared by derivatizing atrazine and simazine and conjugating them via aminohexanecarboxylic acid and thiopropanecarboxylic acid substituents to proteins. Six rabbit antisera and one murine monoclonal antibody were incorporated into the ELISA (enzyme linked immunosorbent assay) format using peroxidase and alkaline phosphatase tracers. Superior antisera for atrazine, simazine and ametryn exhibited in optimized assays a 50% binding inhibition at 0.16, 0.25 and 0.45 μg 1−1, respectively. The IC50 value for the monoclonal antibody to atrazine was about 1.0 μg l−1. The most sensitive antiserum Atr-(CH2)5-OV-3C exhibited 74.1% cross-reactivity with propazine and 26.3% with deethylatrazine. Antisera produced in response to the simazine-S-(CH2)2- determinant showed a remarkable variety in s-triazine recognition selectivity. Antiserum Sim-S-(CH2)2-KLH-C was extremely sensitive to ametryn, terbutryn and prometryn with cross-reactivities based on simazine (= 100%) 2200, 1100 and 1480%, respectively. Structural aspects of these findings are discussed. ELISA for atrazine was evaluated on surface water and ground water samples collected from a contaminated area. The amounts of atrazine found by ELISA were in good agreement with GC-MS analysis. Starting data for the application of ELISA to juice and milk samples are also presented.

Rapid pretreatment-free immunochromatographic assay of chloramphenicol in milk.

A pretreatment-free immunochromatographic assay for detection of chloramphenicol (CAP) in milk was developed. The assay is based on competition between CAP molecules in the sample and immobilized CAP-protein conjugate for binding to monoclonal anti-CAP antibodies conjugated with colloidal gold particles (average diameter 30nm). The assay is carried out in the course of sample flowing along test strip with immobilized reactants, and its results can be detected by the naked eye or by a photometric device. Effect of the concentration of immunoreactants on assay characteristics was studied. The assay protocol with maximal sensitivity and reliability was optimized using measured values of brightness of lines. Detection limit for CAP is 10ngmL(-1). Assay duration is 10min, and it can be carried out at room temperature without any additional devices and reactants. The developed test strip has been applied to CAP detection in dairy products.

Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder.

AbstractA fluorescence polarization immunoassay (FPIA) based on a polyclonal antibody was developed for the determination of melamine in milk. To obtain an antibody with improved sensitivity and specificity, 6-hydrazinyl-1,3,5-triazine-2,4-diamine was coupled to bovine serum albumin and used as the immunogen for the rabbit immunization. Three fluorescein-labeled melamine tracers with different structures and spacer bridges were synthesized. The structural effect of the tracers on the assay characteristics was investigated. 6-(4,6-Diamino-1,3,5-triazin-2-ylamino)-N-(2-(3-(3′,6′-dihydroxy-3-oxo-2,3-dihydrospiro[indene-1,9′-xanthene]-5-yl)thioureido)ethyl)hexanamide demonstrated better sensitivity than 5-(2-(4,6-diamino-1,3,5-triazin-2-yl)hydrazinecarbothioamido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 3-(4,6-diamino-1,3,5-triazin-2-ylthio)-N-(2-(3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-yl)thioureido)ethyl)propanamide. The limit of detection (10% inhibition) of the FPIA was 9.3 ng mL-1 and the IC50 (50% inhibition) value was 164.7 ng mL-1. The antibody in the FPIA showed 21.2% cross-reactivity to the fly-killing insecticide cyromazine, but had no cross-reactivity to other natural structurally related compounds. Recoveries, measured in spiked milk and milk powder samples, ranged from 79.4 to 119.0%. Milk samples fortified with melamine were analyzed by this method and confirmed by high-performance liquid chromatography–mass spectrometry. Excellent recoveries and correlation with spiked levels were observed, suggesting that this immunoassay could be applied to the screening of melamine residues in milk and milk powder after a simple dilution procedure. FigureFigure Performance comparison of tree tracers and two antibodies. (a), antibody A; (b) antibody B

Fluorescence polarization immunoassays for pesticides

Fluorescence polarization immunoassay methods for the detection of pesticides and their metabolites or degradation products are reviewed. Advantages and limitations for application to pesticide detection in environmental and food samples are discussed. The influence of the structure of fluorescent-labeled tracers and the affinity and specificity of antibodies on analytical performance is examined. The methods are simple, readily automated, and rapid (total time for assay of a water sample is about 1 min) with sensitivity of 1 - 10 ng/ml pesticide in 0.01 - 0.1 ml sample.

Fluorescence polarisation for immunoreagent characterisation

Antibodies were characterised using fluorescence polarisation, a homogeneous assay technique in which all reagents are in solution. Kinetic studies on the association and dissociation of the immunocomplex were performed. A competitive assay was used and the sensitivities, operational linearities, as well as the specificities of the immunoassays were experimentally determined for various antibody preparations with specificity for triazines. Detection limits for atrazine in water samples were determined to be within the range of 0.08-0.4 ng ml(-1) using a 5-min incubation time and a 0.5-ml sample volume.

Rapid all-in-one three-step immunoassay for non-instrumental detection of ochratoxin A in high-coloured herbs and spices

A feasible three-step method for ochratoxin A (OTA) rapid detection was developed and applied for OTA screening in high-coloured matrices such as liquorice, ginger, nutmeg, black pepper, white pepper and Capsicum spp. spices at a control level of 10mugkg(-1). The method was based on the clean-up tandem immunoassay column and involved three steps: extract application, washing step and application of chromogenic substrate. A significant simplification of the assay was reached by using an additional frit with conjugate inside the clean-up tandem immunoassay column. The time for analysis was less than 10min, including 5min for colour development. Results were visually evaluated as colour development for negative result or no colour development for positive result. The method was coupled with a simple methanol-based extraction. A total of 27 samples were screened for OTA with the proposed method. It was shown that two samples of red pepper and one sample of liquorice, pili-pili, chilli and cayenne were contaminated with OTA above the control level at 10mugkg(-1), but none of tested ginger, nutmeg, black pepper and white pepper.

Simultaneous determination of multiple (fluoro)quinolone antibiotics in food samples by a one-step fluorescence polarization immunoassay.

This paper describes a rapid one-step fluorescence polarization immunoassay (FPIA) for the simultaneous determination of multiple (fluoro)quinolone antibiotics (FQs) in food samples. Several fluorescent tracers were synthesized and evaluated in the FPIA method based on a broad-specificity of monoclonal antibodies toward FQs. The heterogeneous tracer, SAR-5-FAM, was considered as the optimal choice to prepare the immunocomplex single reagent, which allows a rapid and sensitive displacement reaction by addition of analytes. Optimized single-reagent FPIA exhibited broad cross-reactivities in the range of 7.8-172.2% with 16 FQs tested and was capable of determining most FQs at the level of maximum residue limits. Recoveries for spiked milk and chicken muscle samples were from 77.8 to 116%, with relative standard deviation lower than 17.4%. Therefore, this method could be applicable in routine screening analysis of multiple FQ residues in food samples.

Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals

A monoclonal antibody against zearalenone (ZEA) was produced and used successfully to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) for the analysis of ZEA in cereals. This DC-ELISA had a limit of detection of 0.15 ± 0.02 µg l−1 and an IC50 value of 1.13 ± 0.16 µg l−1. Matrix interference was minimized by dilution of the sample extract before ELISA assays. Aqueous methanol (80%) gave good extraction efficiencies, and the recovery from spiked rice, barley, and corn samples averaged between 87 and 112%. Although ZEA was detected in seven (9%) of 80 rice samples and in eight (16%) of 50 barley samples, the concentration of ZEA in samples was around or below the limit of detection of DC-ELISA. Among 38 corn samples, ZEA was detected in nine (24%) samples in the range 41.0–909.8 µg kg−1. Re-analysis of the ELISA-positive corn samples by high-performance liquid chromatography (HPLC) confirmed that seven (18%) corn samples were positive. The ZEA results for corn showed very good agreement between DC-ELISA and a commercial AgraQant® zearalenone kit (r 2 = 0.98). Thus, the monoclonal antibody-based DC-ELISA could be applied to the preliminary screening of ZEA contamination when analysis of a large sample number is needed.