Elevated sensitivity in an immunoassay using time-resolved fluorescent detection

Abstract: Most commercially available antibody pairs have been designed to be used in sandwich ELISA assays. They offer the flexibility of miniaturizing experiments to 384- and even 1536- well formats for higher throughput demands. Here we use matched antibody pairs (MAPs) which are well-characterized, recombinant antibodies that cover a wide variety of targets. These MAPs consist of an unlabeled capture antibody, a biotinylated detection antibody and include aprotein standard. In order to enhance assay performance, alternative measurement techniques and different secondary anti-body labels can be used. Here we used, Time Resolved Fluorescence (TRF) detection which uses long-lifetime fluorophores, known as lanthanides, that emit light over a longer period after excitation (microseconds as opposed to nanoseconds). This helps reduce background noise by delaying the start of the measurement until after the background signal has decayed. The measurement of assay signal using non-visible light spectra can increase dynamic range and is not limited to the same extent by saturation. Here we describe a 384-well TRF immuno-assay for the detection of IRAK4, a protein kinase involved in cell signaling as part of the immune response. Our results show that this miniaturized assay performs as well as, or better than the traditional approach indicating that it may be possible miniaturize still further and implement this detection in a high throughput setting.