The Detection System in Lateral Flow Assays

Most rapid tests consist of two antibodies—one labeled with a visual tag such as colloidal gold or colored latex, and one used as capture material immobilized on the nitrocellulose membrane. However, with DOA assays, the size of the drug molecule to be detected is relatively small, with perhaps only one immunogenic epitope. In this case, the luxury of a two-antibody detection system does not exist. Therefore, the most common type of rapid test is an inhibition assay. In this type of test, the colloidal gold-labeled antibody is used as the detector reagent, and a drug-carrier protein conjugate is used as the capture material. Whereas the antibody is incorporated into the conjugate pad and is free-flowing, the drug-carrier protein conjugate is immobilized on the nitrocellulose membrane in the form of a narrow line (see Chapter 4). In the absence of drug in a testing specimen, labeled antibodies freely migrate into the nitrocellulose membrane and form antibody-antigen complexes with the immobilized drug-protein conjugates, thereby forming a colored line visualized through the results window. In the presence of drug in the testing specimens, labeled antibodies in the conjugate pad readily recognize and bind to the in-flowing free drug molecules, rendering them saturated in their antigen binding site, and they are are thus unable to bind to the immobilized drug-carrier conjugate when the antibody-drug complexes reach the nitrocellulose membrane. This results in the absence of a colored line in the results window. Note that the appearance of a colored line in the results window correlates with a negative test and the absence of a colored line in the window indicates a positive drug test.

Competition assays where the drug-carrier molecule is conjugated to the label and the antibody is used as the capture agent are also feasible, but are not widely employed. This may be a result of the unlikely success of linking the drug-carrier complex to the label in such a way that the drug is always available to bind to the capture antibody. Any blockade of antibody-antigen binding will undoubtedly result in a lowering of the test sensitivity and possible false-negative results.

Drug detection assays often have different specifications for detection limits depending upon whether they are urine or saliva/sweat based. In addition, the drug metabolite to be detected may be different in each system. It is not necessarily the case that an assay developed for the detection of cannabis in urine, for example, will work satisfactorily with saliva or sweat. Therefore, one must carefully consider the specifications of the assay before progressing to the stage of selecting the biological materials. These types of tests rely on minimal batch variation to achieve the specified level of cut-off consistently. Small variation is inevitable because of the nature of the biological materials used; however, the manufacturer may minimize this variation by choosing quality raw materials.

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