ELISA Assays
Both ELISA tests were performed according to the manufacturer's instructions. The
Atrazine ELISA technology has been previously described by others.(' The test kit used
in this study for the diaminoatrazine ELISA is not currently commercially available.
Beacon Analytical Systems, Inc. of Portland, ME, in conjunction with Syngenta Crop
Protection, Inc., has developed a prototype ELISA for diaminoatrazine. They allowed the
WSLH to use their ELISA kit for this study. The diaminoatrazine test is a competitive
ELISA test method using polyclonal antibodies that bind both diaminoatrazine and
diaminoatrazine-enzyme conjugate molecules attached to the inside surface of test tubes.
Samples of water are added to these tubes along with diaminoatrazine molecules with a
specific enzyme attached (conjugated) and allowed to incubate. During this incubation
period, diaminoatrazine molecules from the sample compete with diaminoatrazine-
enzyme conjugate molecules for a limited number of antibody binding sites on the wall of
the test tube. If there is a lot of diaminoatrazine in the sample, most of the binding sites
capture the diaminoatrazine compound. If there is little diaminoatrazine in the sample,
most of the binding sites are then occupied with the diaminoatrazine-enzyme conjugated
compound. After the incubation period the unbound sample and conjugate are washed
from the antibody coated tubes. This step is analogous to sample extraction and clean up
normally used for chromatography. A substrate solution is then added to the tubes, which
reacts with the enzyme portion of the diaminoatrazine-enzyme conjugate molecule to
form a colored product. If there's a low concentration of diaminoatrazine in the unknown
sample, the binding sites will be filled with the enzyme conjugate molecules and a dark
color will develop. Conversely, if the sample being tested is rich in diaminoatrazine, most
of the sites would be filled with the compound leaving few sites with the enzyme present,
thus resulting in a minimum level of color production. The intensity of the color is
inversely proportional to the concentration of the diaminoatrazine in the sample. Actual
concentrations of the compound can be estimated by comparison to a standard curve.
GC assays
All gas chromatography was performed by the Wisconsin DATCP pesticide laboratory
using standard techniques.(2) Triazine pesticides were extracted from water samples with
methylene chloride followed by ethyl acetate. The extracts were mixed, the solvent was
evaporated to dryness and the sample reconstituted with methyl tert-butyl ether (MTBE).
The MTBE solution was then analyzed by gas chromatography using a nitrogen
phosphorous detector. Confirmation of detections using a different column or detector
was done routinely. Quantification was done by using a calibration curve that bracketed
the concentration of the sample or by peak-to-peak comparison of the unknown to a
standard (whose peak height is ten percent of the unknown peak height). More details on
the method may be found in Method 633 of the DATCP Laboratory Services Manual.(2)