Page:NIOSH Manual of Analytical Methods - 5603.pdf/3

 ALACHLOR in AIR: METHOD 5603, Issue 1, dated 15 January 1998 - Page 3 of 5

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d. Wrap remainder of the samples with parafilm and store refrigerated. e. Run the diluted samples in the assay. ™ Follow the general instructions of the EnviroGard Alachlor plate kit [4]. Adjust as needed to accommodate sample needs. a. Determine the numberof microtiter plate wells needed to run in duplicate the kit negative control and 3 kit standards, the matrix negative control and 3 matrix standards, the samples, and an alachlor fortified control for each set of 3 strips. Remove the number of well strips needed (twelve wells per strip). Refrigerate the unused strips in the resealable plastic bag containing a desiccant provided in the kit. b. Bring the well strips and the remaining contents of the ELISA assay to ambient temperature before beginning the assay. c. Using a pipettor with aerosol resistant tips, in an orderly fashion, add 80 µL aliquots of the standards, samples, and controls to the microtiter plate wells in duplicate. NOTE: The ELISA assay is a time dependent matrix. Add reagents in a consistent, uninterrupted manner to avoid variability and a drift effect over the plate. d. In the same order the standards and samples were added to the plate, add 2 drops of enzyme conjugate from the enzyme conjugate bottle. NOTE: If more than 3 rows of wells are to be employed, use a multiwell pipettor. If the dropper is used, ensure that the drops are free falling and do not touch the sides of the well. e. Carefully mix the contents of the wells with circular motions. f. Cover the plate with parafilm. Incubate for 1 hour at ambient temperature with mechanical mixing on an orbital mixer or microtiter plate shaker. If necessary, sedentary incubation may be used. g. Wash the plate with tap or deionized water 6 times with a microtiter plate washer or manually. If done manually, vigorously flick the contents of the plate into a sink, flood the plate with water, and empty the water with a flick of the wrist. Repeat 6 times. h. Invert the plate over a laboratory towel to remove the excess water. i. As in the previous order of addition, add 80 µL (2 drops) of substrate, and add 40 µL (1 drop) of chromogen from kit. (1). If less than 3 or 4 well strips are used, substrate and chromogen may be added separately. (2). If more, freshly mix a 2:1 volume of the substrate and chromogen, respectively, and add 120 µL of the mixture to each well. NOTE: Use care to prepare sufficient reagent mixture to add to the contents of the wells plus a small reservoir so as not to interrupt the addition of the reagents. Any interruption in the addition of reagents may affect the color development relative to the standard curve. Discard the excess. j. Mix, cover with clean parafilm, and incubate the plate as before for 30 minutes. k. After incubation, add 40 µL (1 drop) of the stop solution (from the kit) to each well, and mix thoroughly until all of the blue has converted to yellow. NOTE: Plate must be read within 30 min after adding stop solution.

CALIBRATION AND QUALITY CONTROL: 7. Check the overall performance of the microplate reader according to the manufacture’s instructions. 8. Prepare a calibration curve, using three standards: 100, 500, 2500 ng/L, each time the assay is run. To prepare the working standards in the appropriate matrix: a. Serially dilute an aliquot of the alachlor stock solution in a volume of matrix similar to the sample matrix yielding a 50,000 ng/L. (If the methanol concentration of the samples is less than 1%, water may be used. However, the higher the methanol concentration of the samples, the more desirable it is to use the same % of methanol standards as in the samples. b. Using the 50,000 ng/L solution, prepare the working standards 100 ng/L, 500 ng/L and 25000 ng/L, in the matrix solvent. 9. Run controls, 1 for every three rows of the assay to determine possible drift. 10. Zero the reader against air or deionized water before reading the assay. 11. Determine the concentration of alachlor using the data reduction capabilities of the microtiter plate reader, or appropriate software. Use a semi-log or 4 parameter logit-log curve fit to the standard curve. Make the appropriate dilution corrections. 12. If data reduction capabilities are not available, perform the calculations as follows: NIOSH Manual of Analytical Methods (NMAM), Fourth Edition