A novel, sensitive, linker-assisted enzyme-linked immunosorbent assay (L'ELISA) was compared to on-line solid-phase extraction (SPE) with high-performance liquid chromatography/mass spectrometry (HPLC/MS) for the analysis of glyphosate in surface water and groundwater samples. The L'ELISA used succinic anhydride to derivatize glyphosate, which mimics the epitotic attachment of glyphosate to horseradish peroxidase hapten. Thus, L'ELISA recognized the derivatized glyphosate more effectively (detection limit of 0.1 microg/L) and with increased sensitivity (10-100 times) over conventional ELISA and showed the potential for other applications. The precision and accuracy of L'ELISA then was compared with on-line SPE/HPLC/MS, which detected glyphosate and its degradate derivatized with 9-fluorenylmethyl chloroformate using negative-ion electrospray (detection limit 0.1 microg/ L, relative standard deviation +/- 15%). Derivatization efficiency and matrix effects were minimized by adding an isotope-labeled glyphosate (2-13C15N). The accuracy of L'EUSA gave a false positive rate of 18% between 0.1 and 1.0 microg/L and a false positive rate of only 1% above 1.0 microg/L The relative standard deviation was +/- 20%. The correlation of L'ELISA and HPLC/MS for 66 surface water and groundwater samples was 0.97 with a slope of 1.28, with many detections of glyphosate and its degradate in surface water but not in groundwater.

Development of an ultrasensitive enzyme immunoassay for the analysis of glyphosate in community water systems.

Author: Mestemacher, Michelle A. ; Bhullar, Balwant S. ; Sangha, Jangbir S.

As an alternative to the currently used slow and complex anal. methods for glyphosate anal., we have developed a rapid, cost-effective and tech. simple ELISA (ELISA) for the quantitation of glyphosate in community water systemsThis assay is based on the principle of direct competitive ELISA where a fixed amount of enzyme-glyphosate conjugate competes against an unknown amount of glyphosate in the test sample for a limited number of antibody binding sites on a solid phaseA simple anion-exchange solid-phase extraction (SPE) step eliminates any matrix effectIn this assay procedure, the standard curve calibrators and the quality control samples are treated exactly the same way as the unknownsPost-column eluates are incubated for 20 min with a proprietary reagent, followed by the addition of horseradish peroxidase-glyphosate conjugateThe assay mixture is then incubated for 40 min in antibody-coated microplate wells, followed by washing and color development for 10 minThe limit of detection of glyphosate for 1-mL size samples is about 50 parts per trillionThis polyclonal antibody-based ELISA is highly specific for glyphosate, showing less than 0.1% cross-reactivity with aminomethylphosphonic acid (AMPA)The total assay procedure, including the pre-ELISA sample preparation steps, can be completed in less than two hours(Patent Pending).

Development of an enzyme immunoassay for the measurement of endothall in environmental water samples.

Author: Mestemacher, Michelle A. ; Bhullar, Balwant S. ; Sangha, Jangbir S.

Endothall is a widely used herbicide with applications in diverse fields, including its use as an aquatic weed control agent in power plants, rivers and lakesCurrently, the endothall levels in ground and surface water samples are being measured by conventional anal. methodsAs an alternative to these methods, we have developed a rapid, cost-effective and tech. simple ELISA (ELISA) for endothallThis assay is based on the principle of direct competitive ELISA where a fixed amount of enzyme-endothall conjugate competes against an unknown amount of endothall in the test sample for a limited number of antibody binding sites on a solid phaseA simple anion-exchange solid-phase extraction (SPE) step eliminates any matrix effectPost-column eluate of each sample is mixed with horseradish peroxidase-endothall, and the assay mixture is incubated for about 40 min in antibody-coated microplate wells, followed by washing and color development for 10 minThe limit of detection of endothall for 1-mL size samples is approx. 5 ppbIf needed, higher assay sensitivity can be achieved by increasing the SPE sample sizeThis polyclonal antibody-based ELISA is highly specific for endothall and its ester derivativesGlucose, glucose-6-phosphate, sucrose, soluble starch, and citric, succinic and malic acids show no cross-reactivity when tested at 1, 10 and 100 ppmThe total assay procedure, including the pre-ELISA sample cleanup/concentration step, can be completed in less than two hours.

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