Development of multiplexed fluorescence microbead covalent assays (FMCAs) for pesticide biomonitoring.
Biagini-RE; Murphy-DM; Sammons-DL; Smith-JP; Striley-CA; MacKenzie-BA
Bull Environ Contam Toxicol 2002 Apr; 68(4):470-477
In the present work we describe FMCAs for A TR and MM, both in the monoplexed and multiplexed format. Atrazine and its metabolites have been measured by high-perfonnance liquid chromatography (HPLC) coupled with tandem mass spectrometry (HPLC-MS/MS) using atmospheric pressure chemical ionization. This method has limits of detection ranging from 20 to 500 ng/l (parts per trillion) and relative standard deviations of less than 11 % (Baker, et al., 2000). Commercially available kits for ATR are available (The Atrazine RaPffi Assay, Strategic Diagnostics Inc., Newark, DE) with a LDD of 0.046 ppb (in water). Commercially available kits for MM analysis are also available (EnviroLogix Metolachlor Mercapturate in Urine Plate Kit, EnviroLogix, Portland, ME) with a LDD of 5 ppb and assay range of 8 - 85 ppb. The LDDs for the FMCAs described in the present work are in the same range with published and commercial LDD ELISA values. The multiplexed FMCAs presented herein have substantial benefits over classical chemical and traditional EUSA methods. The FMCAs are "no-wash" in that sequential reagents are added to the assay without removing the previous reagent(s) by repeated washings. Repeatedly washing of ELISA plates has the possibility of removing analyte as well as affecting the binding of adsorbed antibodies/antigens. In FMCA, the antibodies/antigens are covalently bound to the solid support The FMCAs are considerably faster than ELISAs, having two 30 minute incubations and a 20-40 second assay time (per sample). This speed enhancement may be explained by considerably improved reaction kinetics, the assays being perfonned on the surface of the mobile microspheres vs. immunosorbedladsorbed reagents in immunosorbent assays. The adaptation of the Luminex Flow Metrix system from single tube assays to 96-well plate assays (using the instrument's XY platfonn) adds automated capability to the instrument in that as many as 96 assays can be perfonned in one microwell plate. The ability to multiplex, the savings in time, minimization of systematic error, the "no wash" format and improved reaction kinetics with sensitivity and precision comparable to ELISA methods suggest that multiplexed FMCA for pesticides/pesticide metabolites is a viable adjunct to classical instrumental methods and ELISA methods for urinary biological monitoring.
Acetamides; Herbicides; Urinalysis; Metabolites; Enzymes; Pesticides; Pesticides-and-agricultural-chemicals; Agricultural-workers; Farmers
Division of Applied Research and Technology, National Institute for Occupational Safety and Health, Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, 4676 Columbia Parkway, Cincinnati, OH 45226
Research Tools and Approaches: Exposure Assessment Methods
Bulletin of Environmental Contamination and Toxicology