QUANTITATION OF RT-PCR AMPLIFIED CYTOKINE MESSENGER-RNA BY AEQUORIN-BASED BIOLUMINESCENCE IMMUNOASSAY

We described here a bioluminescence-based immunoassay for the quantita tion of RT-PCR amplified cytokine mRNA. This technique uses a standard RT-PCR procedure, with the following modifications. The forward prime r in the PCR reaction is labeled with a 5' biotin molecule. Following PCR, a digoxigenin-conjugated oligonucleotide probe is hybridized to t he target biotin-labeled DNA template. The hybridized duplex is captur ed onto a streptavidin-coated microtiter plate. The bound product is q uantitated by adding digoxigenin-specific antibodies conjugated with t he photoprotein aequorin. The amount of specific DNA captured onto the plate is quantitated by triggering the bioluminescence reaction throu gh the addition of calcium ions, This technique detected as low as 40 amol of amplified cytokine products, or 500 copies of templates when 2 7 PCR cycles were used, The high sensitivity of this technique enables the quantitation of target DNA during the exponential phase of the PC R reaction. The aequorin-bioluminescence assay is an alterative non-ra dioactive method for the quantitation of PCR products.