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      Development of an Enzyme Linked Immunosorbent Assay and an Immunochromatographic Assay for Detection of Organophosphorus Pesticides in Different Agricultural Products

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          Abstract

          Objective

          Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment.

          Methods

          A broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively.

          Results

          Under the optimized conditions, the IC 50 values of the ELISA ranged from 3.7 to 162.2 ng mL –1 for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC 50 values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL −1. The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides.

          Conclusions

          The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others.

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          Most cited references26

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          Factors affecting the antigen-antibody reaction.

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            Colloidal gold probe-based immunochromatographic assay for the rapid detection of brevetoxins in fishery product samples.

            One-step immunochromatographic assay using colloidal gold-labeled monoclonal antibody (Mab) probe for the rapid detection of brevetoxins (PbTxs) in fishery product samples was developed. The described assay was based on a competitive format using two antibodies. The primary antibody was conjugated with colloidal gold (detector reagent), the secondary antibody (capture reagent) was immobilized within a defined detection zone (control line) on a diagnostic cellulose nitrate membrane. The toxin in sample compete with immobilized toxin to bind with gold conjugated Mab. The mobile complex (colloidal gold-Mab-toxin) can be captured by the secondary antibody but cannot be captured by BSA-PbTx (test line). The color density of the test line correlated with the concentration of PbTx in sample in the range 10-4000 ng mL(-1). Spiked samples were detected by the assay and the visual detection limit was found to be 20 ng mL(-1). This qualitative test based on the visual evaluation of results did not require any equipment. The assay time for PbTx detection was less than 10 min, suitable for rapid testing on-site.
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              Strip-based immunochromatographic assay using specific egg yolk antibodies for rapid detection of morphine in urine samples.

              Using specific egg yolk antibodies (IgY), a strip-based immunochromatographic assay was developed for rapid detection of morphine in urine samples. IgY type antibody against morphine was generated by immunizing chickens with well-characterized monoacetyl morphine-protein conjugate. The antibody was labeled with gold nanoparticles and used as an immunoprobe in the dipstick format for the visual detection of morphine in urine samples. The dipstick was developed using three membranes: an application pad made of glass fiber membrane to hold the tracer, a signal generation test line on nitrocellulose membrane (detection zone) and a cellulose membrane used as an absorption pad. Analytes of interest (morphine and its analogues) added to the sample well, dissolved the labeled antibody (tracer), and the antigen-antibody complex formed was transported by the flow caused by capillary action to the test line. The color signal of test line in proportion to the morphine concentration in urine samples was measured using a detector. The developed dipstick assay format was optimized, showing the average IC(50) values of morphine as low as 9.45 ng/mL, the detection range of 1-1000 ng/mL and the lowest detection limit 2.5 ng/mL under optimal conditions of analysis. The correlation between the developed dipstick and ELISA was 0.948 in the analysis of urine samples spiked with morphine. The developed dipstick could be a highly sensitive and convenient tool for rapid detection of opiate drugs in samples with high degree of stability.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                31 December 2012
                : 7
                : 12
                : e53099
                Affiliations
                [1 ]College of Plant Protection, Nanjing Agricultural University, Nanjing, China
                [2 ]Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
                [3 ]Henan Provincial Key Laboratory for Animal Immunology, Henan Academy of Agriculture Sciences, Zhengzhou, China
                Cordelier Research Center, INSERMU872-Team16, France
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: XH GZ FL. Performed the experiments: XH JY QF. Analyzed the data: XH LW. Contributed reagents/materials/analysis tools: XH JY GZ FL. Wrote the paper: XH FL.

                Article
                PONE-D-12-19722
                10.1371/journal.pone.0053099
                3534045
                23300869
                a787d04f-53d3-41dc-9714-50bec24792c9
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 2 July 2012
                : 23 November 2012
                Page count
                Pages: 11
                Funding
                This work was supported by the National High Technology Research and Development Program of China (2012AA101401-1), the Key Projects in the National Science & Technology Pillar Program (2009BADB9B03), the earmarked fund for the China Agriculture Research System (nycytx-29) and the Natural Science Foundation of Jiangsu Province (BK2010445). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Agriculture
                Agrochemicals
                Pesticides
                Biology
                Immunology
                Immunologic Techniques
                Immunoassays
                Chemistry
                Organic Chemistry
                Organic Compounds
                Organophosphates
                Medicine
                Clinical Immunology
                Immunologic Techniques
                Immunoassays
                Toxicology
                Immunotoxicology

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                Uncategorized

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