Immunoassays performed on chemistry instrumentation housed in the laboratory use antibodies to recognize and bind to specific kinds of drug molecules in a urine sample. A measurable signal is then produced in response to the binding of the drug (the drug serving as the antigen in the immunoassay). Detection of the signal is accomplished with the use of enzyme labels, along with their respective substrates. Examples of enzyme labels include glucose-6-phosphate dehydrogenase (G6PD), horseradish peroxidase, alkaline peroxidase, β-galactosidase and luciferase. The labels on the enzymes can generate several kinds of signals depending on the substrate they are present in. They can emit radiation, produce a color change, fluoresce under light, or they can emit light.
In a competitive immunoassay, a known amount of antibody, labeled-reagent-drug, and substrate are added to a urine sample. The drug (or drug metabolite) in the sample competes with the labeled-reagent-drug for binding sites on the antibody to form antigen-antibody complexes. If there is no drug in the urine sample, the antibody binding sites are available for binding of the labeled drug or metabolite blocking the substrate from reacting with the enzyme. If drug is present in the sample, it would bind to the antibody binding sites, leaving the labeled drug or metabolite available to react with the substrate and illicit a response measured as a change of absorbance by a spectrometer, fluorometer, or luminometer.
