Discussion

Profiling of secreted proteins is ideally achieved by the detection of multiple proteins simultaneously, maximizing contextual and correlative value of the data. However, bead-based multiplex technologies are often slow, complicated and exceed the price per data point goals for screening. IntelliCyt’s Multicyt QBead assays for soluble proteins run on the iQue Screener enable automatable, no-wash, miniaturized, multiplex detection of up to 30 simultaneous analytes, and rapid read times. Based on IntelliCyt’s patented high throughput sampling technology, we are able to sample from 96, 384, and 1536 well plate formats with zero dead volume. Here we demonstrate the miniaturization of our standard QBeads assay for cytokine detection for high throughput screening from a standard 30 µL down to 6 µL total assay volumes in both 384 well low volume plates and 1536 well plates. As a biological model, Jurkat cells were stimulated with a combination of PHA and PMA for 24 hours in batch. A three-plex QBeads assay was performed in triplicate 384 well plates for the detection of IL-2, IL-3 and IL-8. Bead counts per sample averaged ~400 beads (130 beads per plex) with a 7.3% CV across 3 plates, demonstrating high reproducibility in sampling. Quantification of secreted proteins was likewise robust across all 3 plates, with IL-2 being secreted at high levels (2,311 pg/mL, 10.1% CV), IL-8 secreted at moderate levels (853 pg/mL, 8.1%CV) and IL-3 secreted at moderate levels (485 pg/mL, 11.6% CV). In 1536 plate format, average bead count CV was 8.6% for a 3-plex assay, and all other performance metrics were comparable to data seen in 384-well format. Critical factors in the successful execution of QBeads assays in low volume formats were identified and implemented, including methods for control of sample evaporation and edge effects. In summary, the bead input per well represents a greater than 8-fold reduction in reagent consumption compared to other commercially available multiplexed bead products, greatly reducing the price per data point for HTS without any sacrifice to data quality.