Fluorometric Polymerase Chain Reaction (PCR) Enzyme-Linked Immunosorbent Assay for Quantification of Immuno-PCR Products in Microplates

Abstract

Immuno-polymerase chain reaction (immuno-PCR) allows the detection of protein amounts as low as a few hundred molecules. This enhanced sensitivity is useful for a variety of applications in analytical and biomedical sciences. Application of this technique as a routine method requires the rapid quantification of the PCR products, preferably as an automated readout by microplate-based assays. Here, three methods are compared for detecting such amplified products, i.e., direct staining with a fluorescent intercalating dye, an enzymatic assay utilizing doubly hapten-labeled products, and gel electrophoresis. The enzymatic assay, carried out with either chromogenic or fluorogenic substrate for enzymatic signal amplification, was found to be the most sensitive method. The optimized assay was tested in direct immuno-PCR assays for detecting immunoglobulins (IgG) from mouse and rabbit as well as in a sandwich immuno-PCR assay for detecting recombinant hepatitis B surface antigen (rec. HBsAg). Sensitivity limits were found to be as low as 15 fg (10−19mol) IgG, representing a 1000-fold enhancement compared to enzyme-linked immunosorbent assay detection, and about 70 fg (2 × 10−18mol) rec. HBsAg, improving the detection limit of currently available methods by a factor of about 700. The well-reproducible enzymatic amplification signal further enhances the sensitivity of immuno-PCR and should render the method suitable for routine laboratories.