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The page below is a sample from the LabCE course Drug Testing Methods in the Clinical Toxicology Laboratory. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Screening: Immunoassays

Immunoassays use antibodies to recognize and bind to specific kinds of drug molecules (antigen) in a urine sample. A measurable signal is produced in response to the binding of antigen (drug) and antibody that recognizes the drug. This is accomplished with the use of enzyme labels, along with relevant substrates. Examples of enzyme labels are glucose-6-phosphate dehydrogenase (G6DP), horseradish peroxidase, alkaline peroxidase, and β-galactosidase. 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. The most common measuring device for measuring these signals in immunoassay drug testing instruments is a spectrometer.
In an immunoassay, a known amount of antibody, drug or metabolite labeled with enzyme, and substrate is added to a urine sample. The drug or metabolite in the sample competes with the labeled drug or metabolite 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.