Quantifying amplicons with ELISA

Among the numerous assays proposed for quantifying specific nucleic-acid sequences in biological samples, PCR offers the greatest sensitivity and versatility. The assay for quantifying the amount of polymerase chain reaction (PCR) products is a crucial step in any quantitative PCR method. It should be sensitive and specific, able to display a wide dynamic range, nonradioactive, easy to do, and inexpensive. The results of the assay should also be easily digitalized. Quantification of amplicons with enzyme-linked immunosorbent assay (ELISA) fulfills these criteria. It can be automatized and readers are already available in most research and clinical laboratories. This assay can be accomplished by using colorimetry, fluorometry, or luminometry, depending on the substrate used. Luminometry displays the best sensitivity and has the widest dynamic range of these three methods (1 and see Subheading 1.2.3.). In this chapter, we will describe some of the available formats, the one we have been using this past few years, and its use in kinetic quantitative PCR or with internal standard.