January 15, 2016 (Vol. 36, No. 2)

Novel System Enhances Bead-Based Multiplexing Assays Using Less Sample and Reagent While Achieving Optimal Cytokine Sensitivity

Luminex cytokine assays are common bead-based multiplex ELISA assays conducted in 96-well plate formats in 100 µL volume ranges. The theoretical principle of Luminex cytokine assays are based on an analyte binding best to an antibody-conjugated Luminex xMAP® bead using suspension via a “Brownian motion” pattern of mixing. This assay works well in aqueous solutions such as cell supernatants. However, a natural increase in viscosity and complexity of the solution such as bodily fluids (e.g., plasma, serum), limits the ability of beads to mix as randomly and will inevitably lead to some inconsistencies, which will impact results. Furthermore, bead aggregation due to matrix complexity can be a serious problem when beads are agitated and it can invalidate an assay. 

In Curiox Biosystems’ DropArray™ (DA)-Bead Plate method, existing Luminex cytokine assays are performed with few modifications compared to conventional plates. Assays on DA-Bead Plates are essentially prepared with one-fifth of the common conventional reagent volume in a drop on a uniquely designed 96-well wall-less plate.

Importantly, (Figure 1) the beads on the DA-Bead Plate surface are settled and fixed as a monolayer in a drop during incubation with shaking instead of being freely mixed. A magnet maintains Luminex beads as a planar array in the well during shaking. The monolayer of beads on the DA-Bead Plate surface ensures equal exposure of every single bead to cytokines in solution, promoting consistency. The small 15 μL reaction volume, maintained in a drop on the DA-Bead Plate surface, ensures fast diffusion of analytes to the bead surface. Additionally, fixing of beads in DA-Bead Plate prevents the formation of bead clumping.

Washing of Luminex xMAP beads is required in Luminex-based assays to obtain optimal data and sensitivity. The configuration of washing in traditional microtiter plates only allows for dispensing and aspiration of the washing buffer from the top of the well with a pipet or nozzle-based dispenser. Depending on the configuration of washer manufacturers, magnetic arrays are designed to either keep xMAP beads fixed on the side of the well in a highly packed design or spread throughout the surface.

Keeping xMAP beads packed and fixed on a side of the well during washing is prone to generate incomplete washing with a good fraction of beads blocked from exposure to washing buffer in this highly packed configuration. With beads spread on the surface, magnetic configuration is optimal, but nozzle-based washing systems are configured at a high distance from bottom of the well to minimize risk of bead aspiration. As a result, a higher residual amount of wash buffer will be left in the well, leading to incomplete washing.

The DA-Bead Plate washing system is designed to maintain a monolayer of beads during the washing process, thus keeping consistent exposure of every bead to washing buffer. Additionally, DA-Beads’ unique wall-less design enables a washing process using gentle laminar flow, which eliminates residual wash buffer and guarantees complete washing of Luminex beads.

Figure 1. (A) Incubation method principle in a traditional plate (left) and DA-Bead Plate (right). Beads freely floating with shaking in traditional wells at 75 µL volume vs. beads are fixed in a 15 µL drop in DA-Bead Plate by a magnet below the well and shaken. (B) Washing method principle in traditional plate (left) and DA-Bead Plate (right). Magnet array with traditional plate either super-packs beads at the side of a well or keeps beads as a monolayer resulting in aspiration of beads and/or incomplete washing with the nozzle or pipet-based dispenser. Conversely, DA-Bead Plate magnet configuration maintains beads as a monolayer (right), resulting in complete washing with gentle laminar flow and minimal wash buffer leftover.

Comparative Analysis of Cytokine Standards

Performance of Luminex using the traditional method and DA-Bead Plate method can be compared in parallel runs where only 5 μL of beads and 5 μL cytokine standard is needed with DA-beads as compared to 25 μL beads and 25 μL cytokine standard in a traditional run using commercially available reagents. (To view the data, visit curiox.com/GEN)

As an example, we analyzed IL-17F cytokine standard in a serum-based matrix body fluid environment using a traditional method. The curve fit of a traditional plate showed fluorescence saturation around 10 pg/mL of analytes, a relatively narrow LLOQ/ULOQ reliable working range and a limited accuracy of the lowest and highest cytokine dilution points beyond 70–130% ideal range.

Conversely, the curve fit using the DA-Bead Plate method highlights a saturation of fluorescence on a lower level of analytes at ~2 pg/mL, a larger reliable LLOQ/ULOQ working range and good accuracy with the lowest and highest cytokine standard, staying within an accepted 70–130% range in the recovery guidelines. Curve fits from both DA-Bead Plate and microtiter plate methods overlay and correlate well, but overall the Luminex DA-Bead Plate method showed superior performance with IL-17F. 

Overcoming Low Analyte Detectability and Elimination of Bead Aggregation

A group of researchers compared the dataset of 32 mouse plasma samples with a commercially available 25-plex Th17 cytokine panel in a traditional microtiter plate method with 25 μL sample with DA-Bead Plate method with 5 μL sample. As shown, the traditional Luminex method provides a comparative higher number of wells and analytes with no value (out of range) resulting in a low data detectability for IL-21, IL-12p70, IL-4, IL-15, IL-33, IFNγ, IL-31, IL-10, IL-25, IL-23, IL-17F, resulting in an incomplete dataset. When the similar experiment was performed using the DA-Bead Plate method with only 5 μL of the same set of mouse plasma samples, the profile output is significantly improved with an increase in the detectable data for problematic analytes and a fewer number of wells with out-of-range data (Figure 2).

A second group of researchers reports that with certain complex matrix samples, such as rat vitreous extract, the formation of large aggregates of Luminex beads with traditional shaking method during sample incubation. The use of the DA-Bead Plate method confers a drastic improvement in limiting this phenomenon by restricting the ability of beads to aggregate freely in this complex fluid sample. An analysis of the beads behavior by bright field imaging shows how the DA-Bead Plate maintains beads in a linear, monolayer configuration that mimics a planar array format, thereby limiting clogging of the Luminex reader and improving bead count acquisition when compared to the traditional shaking method.

Figure 2. Detectability summary of plasma samples for each analyte. Each bar depicts the percentage of sample well with observed concentration quantity as calculated by Bio-Plex® manager. A well with no value is ranked as OOR (out of range) and was considered not detectable.

Xavier Le Guezennec, Ph.D. ([email protected]), is principal application scientist and Melvin Lye is Manager, Business Development, North America at Curiox Biosystems. Luminex is a registered trademark of Luminex Corporation in the U.S. and other countries.  Luminex Corporation does not endorse Curiox DA-Bead plates.

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