Immunoassays, like ELISA, are performed in plates optimized for protein binding. These are typically made of polystyrene or polycarbonate. For real-time immuno-PCR containers that fit, real-time PCR instruments are needed. Regular ELISA plates do not fit PCR instruments because they have flat bottoms and their size does not match regular PCR blocks. Immunoassays are most conveniently performed in 96-well plates or in strips of eight or twelve tubes. A multichannel pipettor can then be used to add assay components and for washing. Nunc® TopYield polycarbonate strips are flat bottomed and fit normal PCR blocks. These have been used in real-time immuno-PCR attaching the capture antibody by physical adsorption to the wells (Adler et al., 2003). Regular polypropylene plates for real-time PCR, such as the AB-0600 from ABgene, that fit, for example, the iCycler iQ®, from Biorad, can also be used. When we compared the polypropylene plates with the polycarbonate strips we obtained higher reproducibility with the polypropylene container. The polypropylene plates are also available coated with streptavidin for attachment of capture antibody via biotin.
A number of real-time PCR instruments on the market are suitable for real-time immuno-PCR. We find the 96-well platforms most convenient. Our lab has tested the iCycler iQ® from Biorad, the 5700 and 7700 from ABI, the Mx3005p from Stratagene, and the Quantica from Techne, with very good results when using the 96-well microtiter plates recommended by the respective vendors. Also the capillary based LightCycler® has been used for real-time immuno-PCR (McKie et al., 2002). Here the assay was first built up in an ELISA compatible 96-well plate. The DNA-label was then cut by a restriction enzyme and the solution was transferred to LightCycler® capillaries for real-time PCR analysis.
Was this article helpful?