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The suitability of competitive enzyme-linked immunosorbent assays (ELISAs) with chemiluminescent detection-based immobilized antigen (indirect assay) for rapid and accurate determination of chlorpyrifos in various food matrices was tested. The limit of detection (LOD) values were 1–1.75 ng mL−1, the standard curve midpoint (IC50) was 3.5 ng mL−1, and the assay duration was 1.5 h. Assay application to the analysis of honeybee extract resulted in chlorpyrifos recoveries varying between 62 and 83% in 5–15 ng mL−1 herbicide concentration range.

An electrochemical immunosensor based on indirect competitive ELISA technique has been developed and tested for the detection of azinphos‐methyl in aqueous solutions and spiked honeybee extracts. The detection of the pesticide was based on competition for binding to monoclonal antibodies with an ovalbumin (OVA) conjugate, followed by the incubation with anti‐mouse IgG labeled with horseradish peroxidase, whose activity was measured amperometrically with hydroquinone as the substrate. The sensitivity of the azinphos‐methyl assay, estimated as the IC50 value, was found to be 1.2 nmol L−1 (60 min incubation), with a linear range of 0.6–500 nmol L−1 in optimal conditions. The matrix effect on the detection of azinphos‐methyl in honeybee extract was found negligible, with the recovery values in the range 92–105%.

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.

A highly sensitive and rapid chemiluminescent assay for the determination of the activity of xanthine oxidase (XOD) was developed. The chemiluminescent signal was obtained from the catalyzed oxidation of hypoxanthine, accelerated and amplified using a Fe-EDTA complex and perborate, which acts on luminol. The same luminescent mixture was previously used as detection system for immunoassays. Two different mixtures were used, which differ in their luminol and perborate content, with (CLMrho) or without (CLMb) addition of 0.1 μM rhodamine fluorophor. The response obtained from XOD standard solutions in buffer was linear from 5 to 500 U L−1 and from 0.7 to 250 U L−1 for CLMrho and CLMb respectively, at 25°C. 5 and 0.7 U L−1 were the detection limits at 1 standard deviation level. The intra- and inter-assay relative standard deviations ranged from 6 to 12 % for both CLM. Measurements were made using the high performance, low-light level imaging Berthold luminograph LB-980 which allows simultaneous dete...

A highly sensitive and rapid bioluminescent flow sensor was developed for the determination of the content of L-Glutamate (Glu) in serum and stock cubes by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH), produced by immobilized glutamate dehydrogenase (GluDH), with bacterial bioluminescent enzymes immobilized on a separate nylon coil. The response was linear from 50 to 1000 μmol l−1 at 25 °C for the GluDH reactor. The intra- and inter-assay relative standard deviations were less than 7 % and recoveries ranged from 88 to 105 %. The results agreed well with those obtained with a spectrophotometry method for stock cubes and with a chromatographic method for the determination in serum.

A continuous flow bioluminescent method for NADH analysis has been developed using nylon 6 as a solid support. Bacterial luciferase and NAD:FMN oxidoreductase are covalently co-immobilized to a nylon coil (1 m × 1.0 mm ID). The reactor (nylon coil) is placed in front of a photomultiplier tube inside a luminometer linked to an air segmented continuous flow system. Response is linear from 1 to 2500 pmol/tube allowing determination of NADH at picomolar levels. Inter and intra assay precision of the method is satisfactory (5-10%). Range of sample volume injected is 5-100 μl. More than 20 samples per hour can be analyzed and no carryover was observed. The stability of the nylon immobilized enzymes is high (over 2 months) and over 500 samples can be analyzed with a few mg of enzymes.

A continuous flow bioluminescent method for L-alanine analysis in serum and urine has been developed. Serum can be analyzed directly after simple filtration. Response is linear from 50 to 1500 pmoles in biological matrix. Alanine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bioluminescent enzymes. The stability of nylon immobilized enzymes is high (over three months) and more than 900 samples can be analyzed with few mg of enzymes. The results obtained with the bioluminescent sensor agree well with those obtained by ion exchange chromatography (amino acid analyzer).