Unlike traditional immunoassays that detect only single protein analytes in biological samples, bead-based multiplexed assays enable simultaneous detection of multiple analytes in a single well or reaction. They can also provide information about the roles of multiple proteins such as cytokines and other biomarkers that function in diverse biological processes and pathways.
Scientists using multiplexed assays say that compared to planar methods including ELISA, western blotting, and microarrays, multiplexed assays reduce labor, lower costs, and preserve scarce biological samples because multiple analytes can be detected and quantitated in each assay well.
In bead-based assays, immunodetection occurs on polystyrene or paramagnetic microspheres. Each bead has a unique color/spectral address, and each address is associated with a single analyte.
The beads can be further conjugated with a reagent specific to a bioassay, including antigens, antibodies, oligonucleotides, enzyme substrates, and receptors.
In multiplex capture sandwich immunoassays, the capture antibody is coupled to beads of the same color, and the analyte of interest is identified by a detection antibody that is bound to a fluorescent reporter. The use of different colored beads enables the simultaneous multiplex detection of many other analytes in the same sample. A dual detection flow cytometer is used to sort out the different assays by bead colors in one channel and to determine the analyte concentration by measuring the reporter dye fluorescence in another channel.
Multiplexing is accomplished by combining different bead sets with associated capture antibodies into one master mix and incubating that mix with each sample in a microtiter plate. When the assay is read, the reader instrument interrogates each bead. Samples can include plasma, serum, urine, cell culture supernatant, synovial fluid, and cell lysates.
Sample Processing
While each type of sample may require preparation prior to storage or assay, consistent sample preparation and use of established protocols are critical to successful immunoassays, regardless of the sample source.
Prior to running any assay protocol, directions should be reviewed to ensure the necessary equipment is on hand and the role of each step of the protocol is understood. Minimal sample handling is critical to reduce sample degradation and optimize assay results.
Samples should be analyzed shortly after collection or frozen in aliquots. Multiple freeze-thaw cycles of samples should be avoided and all samples should be frozen according to the same protocol.
Sample thawing at 2–8°C is advised when samples are taken from frozen conditions; samples should be maintained in the same temperature range until ready for use. Adequate sample mixing in a vortex mixer for about three seconds at medium speed will eliminate gradients in protein levels that often form with storage. Some samples such as tissue homogenates may require clarification by ultracentrifugation due to high debris levels. Other more viscous samples such as cyst fluid will require dilution in appropriate buffers to allow for assay beads to move freely in the assay mixture.
Sample Pipetting
Samples should be pipetted and aliquoted during the assay procedure using polypropylene pipette tips. Polypropylene helps prevent protein adherence to the pipette tip to ensure more accurate assays; the use of polypropylene assay plates is also recommended.
Those samples with concentrations that exceed the standard curve should be diluted and reanalyzed. Serum or plasma samples should be diluted in assay diluent; tissue culture supernatants should be diluted in the corresponding tissue culture medium.
Maintaining Sample Integrity During Assay
When pipetting samples into wells, it is important to avoid sample cross-contamination with buffers. We recommend adding the samples as the last assay component to the plate wells.
Once pipetted into the wells, plates should be mixed to ensure contact between the sample and assay components. The plate shaker should be set at a speed that ensures thorough mixing without allowing well contents to splash out of the plate.
Sample Additives or Matrix Effects
Matrix effects are caused by fluctuations in the reactivity of the analyte due to variations in its environment in the sample. Immunoassays often exhibit sensitivity to the matrix due to effects on antigen-antibody binding, efficiency of separation of bound and unbound fractions, and the extent of nonspecific binding. Differences among sample matrices and the effects they may exert on assay results should be considered. For example, serum has lower levels of protein—3–4% less due to the removal of fibrinogen from the clotting process—whereas plasma has more protein as anticoagulants are still present when processed from blood. Some anticoagulants, in particular heparin, may also absorb certain cytokines.
While difficult to recommend universal conditions for all the assays of a given panel, sticking with one matrix helps with data continuity.
Conclusion
Although ELISAs remain the gold standard of immunoassays, measurement of only a single protein in each sample severely constrains the amount of information obtained from limited sample amounts such as biopsy material. Commercially available multiplex technologies that can detect large numbers of proteins in a limited volume now offer investigators opportunities to begin addressing complex responses, for example inflammatory responses using cytokine measurement.
As with other immunoassays, robust and consistent sample-preparation methods are critical to ensure accurate and reproducible data and successful multiplexed immunoassays.
Anna Lokshin is associate professor of medicine and pathology at the University of Pittsburgh. She is also director of the Luminex Core Facility of the University of Pittsburgh Cancer Institute.
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