ASSAY PRINCIPLES

The enzyme immunoassay for atrazine is based on the competition between the atrazine to be assayed and the Atrazine-Alkaline Phosphatase conjugate, for binding to rabbit antibody directed against atrazine, coated onto microwells. The sample containing the atrazine, and the Atrazine-Alkaline Phosphatase conjugate, when added to the microtiter wells, compete for binding to a limiting number of antibody sites. After incubation, each well is rinsed to remove non-bound components. The bound enzymatic activity is then measured by the addition of a chromogenic substrate. The intensity of the color developed is inversely proportional to the concentration of atrazine in the sample. The concentration is calculated based on a standard curve. The Atrazine Plate Kit uses polyclonal antibodies that bind both atrazine and an atrazine-enzyme conjugate. Any atrazine present in the sample competes with an atrazine-enzyme conjugate for a limited number of antibody binding sites. Antibodies, which bind atrazine, are immobilized to the inside of the test wells. In the assay procedure, you will:

•   Add samples or calibrators containing known amounts of atrazine and an atrazine enzyme conjugate to test wells coated with anti-atrazine antibodies.
•   Wash away any unbound molecules, after you incubate this mixture for 60 minutes.
•   Add clear substrate solution to each Plate. In the presence of bound atrazine-enzyme conjugate, the substrate is converted to a blue compound. One enzyme molecule can convert many substrate molecules.

Since the same number of antibody binding sites are available in every well, and each well receives the same number of atrazine-enzyme conjugate molecules, a sample containing a low concentration of atrazine allows the antibody to bind many atrazine-enzyme conjugate molecules. The result is a dark blue solution.

Conversely, a high concentration of atrazine allows fewer atrazine-enzyme conjugate molecules to be bound by the antibodies, resulting in a lighter blue solution.