Citation:

CHUANG, J. C., J. M. VAN EMON, J. DURNFORD, AND K. W. THOMAS. AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) METHOD FOR THE URINARY BIOMONITORING OF 2,4-DICHLOROPHRENOCYACETIC ACID (2,4-D). Presented at International Society of Exposure Analysis Conference, Tucson, AZ, October 30 - November 03, 2005.

Impact/Purpose:

The overall objectives of the task include several components: (1) develop immunochemical methods for compounds difficult to analyze by conventional methodologies; (2) tailor immunochemical methods to support specific human exposure assessment studies; (3) team immunochemical sample preparations with instrumental analysis such as mass spectrometry for in-depth sample characterization; (4) provide methods to support NERL's human exposure and environmental monitoring efforts; (5) provide analytical methods that improve risk assessments by reducing the amount of uncertainity in environmental measurements; (6) provide multimedia analytical methods to support an integrated multimedia approach to assess and characterize risk to human health and the environment; (7) provide sponsorship of annual immunochemistry research meetings as a forum for stimulating interest and discussion on current or emerging bioanalytical methods; (8) develop and incorporate rapid, cost-effective laboratory and field portable immunochemical techniques such as enzyme-linked immunosorbent assay (ELISA) methods into monitoring studies and human exposure field surveys to delineate sub-populations of "highly exposed" individuals for detailed follow-up studies.

Specific method needs have been identified through consultations with client office personnel. This Task strives to fulfill those needs as appropriate. In particular, methods for pyrethroids, e.g., permethrin, cypermethrin, and deltamethrin are being developed and evaluated. Efficient sample preparations are under development for exposure samples using pressurized liquid extraction. Confirmation will be achieved using high performance liquid chromatography (HPLC). A rapid immunoassay approach for the analysis of 2,4-D in urine will be completed and a SOP and report written. Immunoaffinity chromatography sample preparations for the pyrethroids will be developed. Work will continue on the application of antibody replacements such as molecularly imprinted polymers. Additional candidate analytes will be identified for a tiered approach and to guide development of the next Task. Flexibility will be maintained to address methods and measurement issues as they arise during the task period which ends in FY06. The objective of the Task is to develop bioanalytical methods to support exposure monitoring studies during the task period.

Description:

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline, 0.05% Tween 20, with 0.02% sodium azide, and analyzed by a 96-microwekk plate immunoassay format. No clean up was required as a dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA Pesticide Exposure Study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichlorometane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%RSD) of the

96-microwekk plate triplicate analyses ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within 120%. Quantitative recoveries of 2,4-D were obtained for the spiked urine samples by the ELISA method (>70%) as well as by the GC/MS procedure (>80%). The overall method precision of these samples was within 120% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30 ng/mL for the streamlined ELISA method. The ELISA and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Results indicated that the ELISA method can be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.

The US EPA through its Office of Research and Development funded and managed the research described here under EPA Contract 68-D-99-011 to Battelle. Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy. Mention of trade names or commercial products does not constitute an endorsement or recommendation for use.

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