Development of Time-Resolved Fluoroimmunoassay for Detection of Cylindrospermopsin Using Its Novel Monoclonal Antibodies.

Cylindrospermopsin (CYN) is a cyanotoxin that is of particular concern for its potential toxicity to human and animal health and ecological consequences due to contamination of drinking water. The increasing emergence of CYN around the world has led to urgent development of rapid and high-throughput methods for its detection in water. In this study, a highly sensitive monoclonal antibody N8 was produced and characterized for CYN detection through the development of a direct competitive time-resolved fluorescence immunoassay (TRFIA). The newly developed TRFIA exhibited a typical sigmoidal response for CYN at concentrations of 0.01⁻100 ng mL−1, with a wide quantitative range between 0.1 and 50 ng mL−1. The detection limit of the method was calculated to be 0.02 ng mL−1, which is well below the guideline value of 1 μg L−1 and is sensitive enough to provide an early warning of the occurrence of CYN-producing cyanobacterial blooms. The newly developed TRFIA also displayed good precision and accuracy, as evidenced by low coefficients of variation (4.1⁻6.5%). Recoveries ranging from 92.6% to 108.8% were observed upon the analysis of CYN-spiked water samples. Moreover, comparison of the TRIFA with an ELISA kit through testing 76 water samples and 15 Cylindrospermopsis cultures yielded a correlation r² value of 0.963, implying that the novel immunoassay was reliable for the detection of CYN in water and algal samples.

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.

[The use of ultrasensitive time-resolved fluoroimmunoassay for rapid detection of microcystin LR].

OBJECTIVE: In order to provide a rapid and selectivity method for the determination of microcystin LR (MCLR). METHODS: An indirect competitive time-resolved fluoroimmunoassay (TRFIA) was developed. MC-LR-BSA was coated by physical adsorption onto the microtitre plate, MC-LR or sample with MC-LR as a competitor. Both them were incubated with limited anti- MC-LR antibody. and a goat antirabbit IgG-Eu3+ conjugate was used as a tracer. RESULTS: The sensitivity of MC-LR-TRFIA was 0.01 microg/L, and the recovery rate was 99.7%. RSD of CBL-TRFIA were 3.9%. The sensitivity of MC-LR -TRFIA provided a linear reaponse from 0.01 - 20 microg/L, with ED50 of 0.57 microg/L or ED80 of 0.16 microg/L and ED20 of 1.70 microg/L. The cross reactivity of the MC-LR-TRFIA with MC-LF and MC-RR was 0.73% and 35%. Both MC-LR-TRFIA and MC-LR-ELISA test were applied for the quantitative measurement of MC-LR in the same samples, and the coefficient of correlation was 0.958. CONCLUSION: The method is suitable to determine MC-LR in water samples.

[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.

Work-related symptoms and Salmonella antibodies among wastewater treatment plant workers.

Wastewater treatment plant workers are exposed to microbes, including Salmonella, but the prevalence of antibodies against Salmonella species or serovars in their serum samples has not been studied. Antibodies against Salmonella Infantis and lipopolysaccharide antigen common to S. Enteritidis and S. Typhimurium in immunoglobulin classes IgA, IgM and IgG were measured from 79 serum samples of wastewater treatment plant workers and from 79 blood donor samples. Faecal samples for Salmonella and Campylobacter were studied. Gastrointestinal, dermal and other symptoms were compared between 81 wastewater treatment plant workers and 89 food-processing workers. The blood donors had more antibodies against all of the tested antigens expect for S. Infantis in IgM and IgA classes, even though the wastewater treatment plant workers had more gastrointestinal symptoms than the controls. No Salmonella or Campylobacter were found in any faecal samples. Salmonella is not a probable cause of symptoms among wastewater treatment plant workers.

Role of the coypu (Myocastor coypus) in the epidemiology of leptospirosis in domestic animals and humans in France.

The coypu (Myocastor coypus), a rodent whose natural habitat is stagnant freshwater, has become a widespread pest in France within the last decade. This study investigated the prevalence of seropositivity and the renal carriage of leptospires in coypus in order to evaluate their role in terms of the risk of infection by Leptospira interrogans in domestic animals and humans. The study involved the application of serological and bacteriological methods to identify leptospires infection and/or carriage in 738 coypus trapped from 1996 to 1999 in six areas of France. Seroprevalence in samples ranged from 16.5 to 66%, and three field strains were isolated (two L. interrogans Icterohaemorrhagiae and one L. interrogans Sejroe). This first report on the isolation of leptospires from coypus in France emphasises the role of this animal in the epidemiology of leptospirosis.

Bacterial surface display of an anti-pollutant antibody fragment.

A peptidoglycan-associated lipoprotein (PAL) fused to an antibody fragment (scFv) specific to the herbicide and environmental pollutant atrazine, has been successfully targeted to the cell surface of Escherichia coli. Anti-atrazine binding could be observed via an atrazine-alkaline phosphatase conjugate. Cells containing the PAL fusion grew with little cellular toxicity when compared with the control. In contrast, expression of anti-atrazine antibody fragments alone caused the cells to lyse after 4 h. The surface display of anti-pollutant antibodies may have a future role in the bioremediation of contaminated water or the development of pollutant-specific, whole-cell biosensors.