Chemiluminescence

Radioactive reagents have been gradually replaced by nonisotopic reagents for some tasks in molecular biology. Concern over laboratory safety and the economic and environmental aspects of radioactive waste disposal have been key factors in this change. Generally, the new nonisotopic systems have improved in terms of analytical sensitivity and the time required to obtain a result. The most prominent nonisotopic analytical methods exploit chemiluminescence, described here. This technique has been particularly effective when used in combination with an enzyme label, so that the amplifying properties of an enzyme label and the high sensitivity of a chemiluminescent detection reaction are combined to produce an ultrasensitive assay (e.g., chemiluminescent detection of peroxidase- and alkaline phosphatase-labeled proteins and nucleic acid probes). In all of the commonly used applications in molecular biology, the analytical performance of the chemiluminescent systems approaches that of ¹²⁵I- or ³²P-based systems. Chemiluminescent systems also avoid the lengthy signal detection times required with ³²P-based methods, yielding results in minutes rather than days. In addition, chemiluminescent probes can be easily stripped from membranes, allowing the membranes to be reprobed many times without significant loss of resolution. Experimental protocols for directly attaching nonisotopic labels to nucleic acids and indirect labeling methods based on biotin, fluorescein, and digoxigenin labels are now well established. The ancillary reagents (e.g., avidin, streptavidin, antidigoxigenin, and antifluorescein enzyme conjugates) required for the indirect methods are widely available.