April 15, 2015 (Vol. 35, No. 8)

Joseph Zock Senior Director Intellicyt
Kim Luu, Ph.D. Director Intellicyt

Detection of Highly Multiplexed Assays in 6 μL Sample Volumes

Choosing assays for pharmaceutical screening is often a compromise between using the ideal target biologies and the harsh realities of reagent availability and cost. This is especially acute during secondary and tertiary screening where creating a multi-faceted picture of potency, specificity, bio-availability, and toxicity is key to choosing lead compounds with the highest chance of clinical success.

An added complexity is the desire for assays to move to a more physiologically relevant state by using primary cells, often in mixed cell populations, and to multiplex assay outputs in order to get as much information as possible from the limited supply.

In many cases limited access to enough of the right cells and/or the cost of reagents prohibits the use of many important biological assays that could be used to make better decisions earlier. Suspension cell and bead assays detected by standard flow cytometry are particularly costly due to the “dead volumes” required in each well to get the biology to the detector. Multiplexing can exacerbate the problem, driving the cost per well to financially intolerable levels.

One well known example is cytokine profiling from primary peripheral blood mononuclear cells (PBMCs), where many of the current multiplexing assay formats can be many dollars per well, which typically relegates these assays to very late in the drug discovery process.

IntelliCyt has developed a solution to overcome this barrier by enabling dramatic miniaturization of many suspension cell and bead assays while retaining assay performance and the ability to multiplex. The key enabling feature for assay miniaturization is the ability to sample as little as 1 µL per sample with no dead volume, allowing dramatic reduction of sample volumes and much lower critical reagent usage.

IntelliCyt’s MultiCyt® screening reagents provide validated, no-wash solutions for multiplexing, enabling the detection and quantification of up to 35 simultaneous analytes and cell-based endpoints per sample.  MultiCyt screening reagents coupled with the iQue® Screener’s patented technology enables 384 well plates to be automatically sampled, analyzed, and visualized in as little as 15 minutes. Cell and/or bead measurements are collected at a rate of up to 10,000 events per second, and a seven decade dynamic range coupled with hardware-based artifact rejection enable no wash assay formats for many applications.

Cytokine Profiling

Here we present a case study of cytokine profiling where our robust, high-throughput profiling platform was used to miniaturize multiplexed bead based assays down to 6 µL total assay volumes in both 384-well low volume and 1,536-well plates. Successful miniaturization of the MultiCyt QBeads assay was facilitated by the fact that the reagent kits were developed as validated no-wash screening protocols. The reagent addition volumes were decreased from 10 µL at each step to 2 µL, without any further alteration to the protocol (Figure 1). 

Of critical importance to the robustness of sampling from low volume plates is the accuracy of the system calibration. All studies were conducted on systems equipped with a GenII probe, which facilitates contact sensing and automated plate mapping. This probe configuration is now standard for both the iQue Screener and iQue Screener HD.

 Another key factor in the robustness of running low volume assays is the ability to sample from sealed plates to prevent evaporation from the wells. A pierce-able heat seal composed of 20 µm thick foil was selected as optimal. Seals composed of different materials (fibrous mats, etc.) can create sampling artifacts, and likewise adhesive seals are not recommended as the adhesive has the potential to damage the sampling capability of the system.


Figure 1. iQue Screener and MultiCyt reagents enable a miniaturized, no-wash assay workflow. QBeads enable a simplified, no-wash workflow on IntelliCyt Screening Systems. (1) 2 µL of beads are incubated with 2 µL of sample for 1 hour. (2) 2 µL of detection reagent is added then incubated for 2 hours. (3) The plate is sealed and read directly on an iQue Screener (either standard or HD). Plate reads typically take less than 20 minutes for 384-well plates and ~1 hour for 1,536-well plates, regardless of the number of analytes multiplexed for detection.

The performance of QBeads in 384-well low volume plates was demonstrated using a biological model of Jurkat cells stimulated with 1 μg/mL phytohemagglutinin (PHA) and 25 ng/ml PMA for 24 hrs. This model is known to promote the secretion of cytokines, including IL-2, IL-3, and IL-8. A 3-plex QBeads assay was performed in triplicate plates to quantify the secretion of IL-2, IL-3, and IL-8 (n= 144 per plate). Results were quantified by the use of standard curves on each plate.

Figure 2 shows the results of the quantified cytokine secretion for the analytes, across all three plates. All analytes were within the published ranges for detection, and demonstrated consistency across the assay. The results for quantity and plate-to-plate %CV for the negative samples (un-stimulated Jurkat cells) were below the assay limit of detection and are not reported. The plate to plate %CV for the positive samples (stimulated) was ~10% for all three analytes.

For the QBeads assay run in 1,536-well plates, we directly compared results from the 6 µL assays to results from 384-well plates using the standard assay volume of 30 µL.  A 3-plex mixture of QBeads standards (IL-2, IFN-γ, and TNF) was tested in 12-pt dose response in replicate across an entire 1,536 well plate (n=128 replicates), and the lowest detectable test concentration greater than 3 standard deviations above negative control for each analyte was calculated.


Figure 2. Detection of cytokine secretion from stimulated Jurkat cells. 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 three plates, demonstrating high reproducibility in sampling. Quantification of secreted proteins was likewise robust across all three plates. Cytokine secretion in negative supernatants were below the assay limit of detection and are reported as ND.

Figure 3 presents individual dose response shown by analyte. Each plot represents the mean and standard deviations across 11 points, with the 12th point being the zero concentration control. Values for the 6 µL no wash assay are shown in comparison to the published specifications for these analytes in standard volume (30 µL) multiplexed format. IFN-γ demonstrated a similar detection limit, and TNF and IL-2 yielded slightly lower detection limits than the published specification.

A representative heat map showing the median fluorescence intensity values used to calculate IL-2 concentrations graphically shows the reproducibility of the dose response across the plate. This representation also provides confidence that the assay is not experiencing edge effects due to evaporation or other signs of sampling and assay artifacts.


Figure 3. Reproducibility of QBeads standards in 1,536-well plates and reproducibility of IL-2, IFN-?, and TNF protein standards. Error bars show standard deviations (n = 128 at each point). The lowest detectable test concentration is comparable to data generated in 384-well standard volume assays.

Conclusions

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 multiplexing technologies are often slow, complicated, and exceed the price per data point goals for screening. 

The iQue Screener and iQue Screener HD are optimized solutions for multiplexed, high-throughput screening of suspension samples—including cells and beads. In this work 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 reduction in volume and correlating reduction in bead input results in cost savings of 8 to 16 fold compared to other commercially available multiplexed bead products with no sacrifice to data quality. 

Kim Luu, Ph.D. (kluu@intellicyt.com), is director of assay development, and Joseph Zock (jzock@intellicyt.com) is senior director of product management at IntelliCyt.