[Immunosensor for rapid detection of 1,3-dinitrobenzene].

An immunosensor for the rapid detection of 1,3-dinitrobenzene was developed based on an evanescent wave all-fiber biosensing platform with the detection limits of 0.054 mg x L(-1), and the detection cycle was less than 10 min. Hapten-carrier conjugates NB-OVA were synthesized by mixing 4-nitrohippuric acid and OVA activated by EDC, and then the conjugates were immobilized onto the silane layer on the probe with a heterobifunctional crosslinker. The probe modified had good robustness and regeneration performance, which allowed the performance of more than 100 assay cycles without significant loss of reactivity. Several water samples of different origins were measured with less than 4.5% -10.0% deviation of the detection and the recovery rate of 1,3-dinitrobenzene was between 80% and 120%, which proved the system's precision and accuracy and negligible matrix effects. This immunosensor shows great potential in rapid detection of 1,3-dinitrobenzene in practical waters.

[Matrix effect and control of immunoassay for environmental samples].

Immunoassay provides very specific, highly sensitive, rapid, and cost-effective analyses for a variety of environmental contaminants. Since the immunoassay detects the environmental samples without pre-treatment, the interferences caused by various matrixes of environmental samples are a major problem, which can greatly affect the detection results. In this paper, based on the enzyme-linked immunosorbent assay (ELISA) for detection of Microcystin-LR (MC-LR), the effect of many kinds of matrixes on ELISA was systematically analyzed, and the corresponding method to control or eliminate the disbennifit effect was proposed. Finally, an integrated and packaged strategy-using the following buffer system as a diluent, i.e. 0.1 mol x L(-1) pH 7.4 PBS (10 x PBS) containing 1% BSA, 0.5% EDTA and 40 g x L(-1) sodium chloride-was given to control the overall matrix effects of environmental samples on ELISA detection. This strategy is also applicable to other kinds of immunoassays for environmental samples.

Matrix Effects on the Microcystin-LR Fluorescent Immunoassay Based on Optical Biosensor.

Matrix effects on the microcystin-LR fluorescent immunoassay based on the evanescent wave all-fiber immunosensor (EWAI) and their elimination methods were studied. The results indicated that PBS and humic acid did not affect the monitoring of samples under the investigated conditions. When the pH was less than 6 or higher than 8, the fluorescence signals detected by immunosensor systems were obviously reduced with the decrease or increase of pH. When the pH ranged from 6 to 8, IC(50) and the linear working range of MC-LR calculated from the detection curves were 1.01∼1.04 µg/L and 0.12∼10.5 µg/L, respectively, which was favourable for an MC-LR immunoassay. Low concentrations of Cu(2+) rarely affected the detection performance of MC-LR. When the concentration of CuSO(4) was higher than 5 mg/L, the fluorescence signal detected by EWAI clearly decreased, and when the concentration of CuSO(4) was 10 mg/L, the fluorescence signal detected was reduced by 70%. The influence of Cu(2+) on the immunoassay could effectively be compromised when chelating reagent EDTA was added to the pre-reaction mixture.

A highly specific immunoassay for microcystin-LR detection based on a monoclonal antibody.

Microcystins (MC) are cyanobacterial hepatotoxins responsible for animal-poisoning and human health incidents. Immunoassays provide a sensitive and fast means to detect these toxins, but cross-reactivity (CR) characteristic of different antibodies was variable. Here, we have produced and characterized a monoclonal antibody (Clone MC8C10) with highly specificity against the most frequent and most toxic variant of microcystins, MC-LR. MC8C10 is more specific against MC-LR among the reported antibodies before. The immunogen was synthesized from the modified MC-LR and bovine serum albumin (BSA). An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with MC8C10 was established to detect the MCs in waters, which showed highly specificity with MC-LR, and have a detection limit for MC-LR 0.1 microg L(-1), the 50% inhibition concentration (IC50) for MC-LR was 1.8+/-0.1 microg L(-1) and the quantitative detection range was from 0.3 to 10 microg L(-1). The [4-arginine] microcystins and the nodularin-R showed lower cross-reactivates (CR<10%), and other MCs such as MC-LF and MC-LW are not recognized (CR<10(-4)). The analysis results of real water samples with ic-ELISA showed that all the coefficients of variation were less than 15%, and the recovery was (100.3+/-5.9)%. So the highly specific ic-ELISA will commendably suit for sensitive analysis for MC-LR in surface water as well as drinking water.

[Detection of microcystin-LR in waters using indirect competitive ELISA].

Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established to detect microcystin-LR in waters, with the concentration of the complete antigen was 5microg/mL, the dilution of the monoclonal antibody was 1:3,000, the dilution of the enzyme tracer (goat anti-rabbit IgG-peroxidase) was 1:3,000, the concentration range of microcystin-LR was between 0.001 approximately 30microg/L, and using o-phenylenediamine as substrate. The assay showed a high relativity of more than 99% with high performance liquid chromatography, a mean relative standard deviation less than 10% , a detection limitation under 0.01microg/L and quantitative detection range was 0.01 approximately 3microg/L, high specificity for [4-arginine] microcystin, and it could still perform well under the influence from the samples.

[Preparation of polyclonal antibody against microcystin-LR].

Good quality polyclonal antibody against Microcystin-LR (MC-LR) was obtained from the New Zealand rabbit immunized with the homemade complete antigen BSA-MC-LR. Indirect ELISA shows the titer of this antibody is more than 1.5 x 10(5); Indirect competitive ELISA immobilized the coating antigen OVA-MC-LR was adapted to detect MCs, the calibration curve shows that the detection lower limit is 10 ng/L, and the range of quantitative detection was between 30 ng/L to 3 microg/L, with a good linearity, which will well potentially suit for sensitive analysis for MC-LR in drinking as well as surface water samples.

A comprehensive immunoassay for the detection of microcystins in waters based on polyclonal antibodies.

Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria (blue-green algae), and microcystin-leucine-arginine (MC-LR) is among the most frequent and most toxic microcystin congeners. In this study, a free amino group was introduced to MC-LR at its seventh amino acid residue with 2-mercaptoethylamine, and the product aminoethyl-MC-LR was coupled to bovine serum albumin (BSA) and horseradish peroxidise (HRP) by glutaraldehyde to be complete antigen (MC-LR-BSA) and labelled hapten (MC-LR-HRP), respectively. Polyclonal antibodies against MC-LR were generated by immunization with MC-LR-BSA. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was established to detect the MCs in waters, which showed a good cross-reactivity with MC-LR, MC-RR, MC-YR, MC-LF, MC-LW and nodularin, and have a detection limit for MC-LR 0.12 microg L(-1), the 50% inhibition concentration (IC50) for MC-LR was 0.63+/-0.06 microg L(-1) and the quantitative detection range was from 0.17 to 2.32 microg L(-1), the analysis result of water samples showed good recovery and reliability. So the comprehensive and reliable dc-ELISA will well potentially suit for sensitive analysis for total MCs in drinking as well as resource water samples.

[Synthesis of complete antigen of microcystin-LR and its antibody production].

Based on the analysis of coupling location, coupling regent, carrying protein and coupling process, completed antigen of Microcystin-LR (MC-LR) was synthesized. A free amidogen was introduced to microcystin-LR (MC-LR) using chemical modification (aminoethylation) of its 7th core amino acids, N-methyl-dehydroalanine. The intermediate product Aminoethyl-MC-LR (H2N-etMC-LR) was purified by Solid Phase Elute (SPE) and identified by Mass Spectrometry, then coupled with BSA through glutaraldehyde to be complete antigen. The completed antigen was dialyzed and identified by SDS-PAGE, UV-scanning and bio-mass spectrometry. The result show that the coupling ratio between MC-LR and BSA of complete antigen in average was over 5, and it is sufficient to immunity.