Multiplex Suspension Array Research Articles

Variance in multiplex suspension array assays: intraplex method improves reliability

BackgroundFlow cytometry based suspended microarray assays are susceptible to many sources of variance; multi-well replication and inter-instrument reproducibility is uncertain.Method and resultsAn "intraplex" method was developed in order to minimize differences in sample readings between instruments. A full intraplex assay consists of a set m of microparticle set classifications assaying for the same analyte, with each of the m classifier sets having different sensitivity to analyte, and n classifier sets replicating each of the m levels of sensitivity, where m > 1 (generally m > 4 would be used).ConclusionThe intraplex method can compensate adequately for the sources of variance that have been identified in suspended microarray assays. It requires no changes to current equipment in use, and is a superior method of constructing precision assays. Additionally, Luminex® users may want to consider the evidence that shows that despite calibration to the same standard, two instruments may not give similar results for all concentrations of analytes.

Parallel processing in microbiology: Detection of infectious pathogens by Luminex xMAP multiplexed suspension array technology

Multiplexed testing — simultaneous analysis of multiple analytes from the same sample — is rapidly becoming a standard in research and clinical laboratories. Drivers for multiplexing include cost savings, labor efficiencies, sample conservation, and work flow efficiencies. Multiplexing can often also be a better approach to understanding the complex biology that frequently underlies disease, and no better example can be found than in diagnostic microbiology, where similar outward signs and symptoms can be manifest from a diversity of infectious pathogens. In this report, we describe representative multiplexed testing applications of the Luminex xMAP system, which couples bioassays with advanced digital signal processing and proprietary identification techniques to perform multi-analyte testing of up to 100 features in real time. The general configuration of an xMAP assay can be described as a suspension array where specific capture moieties are covalently coupled to the surfaces of internally dyed microspheres. Fluorescent dyes contained within the microspheres provide unique spectral characteristics, allowing each microsphere set to be distinguished from all others in the multiplex. The target is labeled for fluorescent detection. Benefits of the system include speed, economy, flexibility, and advanced capabilities. The potential for simultaneous detection of tens, hundreds, and even thousands of protein and nucleic acid targets provides for simultaneous, rapid, sensitive, and specific molecular analyses. A variety of commercial applications are available for the platform, including FDA-approved 510k submissions, but the system is built on an open architecture that allows the assay developer to “home brew” applications as well. In this report, we describe examples of rapid, multiplexed protein and nucleic acid analyses for infectious diseases that demonstrate the utility of the platform.

Variance in multiplex suspension array assays: carryover of microspheres between sample wells

BackgroundThis study was undertaken because of the accidental observation that a sample of 60+ beads was obtained by the instrument from a completely dry, unused well in a 96 well plate. Others have observed unexplained outliers in replicated wells. The problem was first observed on an older instrument, and replicated on a new instrument.Methods and resultsData is presented from two instruments using a multiple blank following well experiment that shows a surprising amount of carryover that has an unexpected nature. When it occurs, it does not necessarily decline from one well to the next. There appears to be two types of carryover, one that is small, predictable and declines consistently, and another which is potentially very large, unpredictable, and does not decline. The former can be compensated for or ignored. The latter cannot be addressed without using multiple replicated samples or an intraplex method.ConclusionThis problem has significance for analysis of results obtained with suspended microarray instruments. A special notation is made that biostatisticians need to be made aware of these results before experiments are undertaken and data generated for them to analyze. The problem can be handled by enough replicated samples, or an intraplex method. The applicability of these results to oligonucleotide based assays is unknown.

Polymorphisms in the glucocerebrosidase gene and pseudogene urge caution in clinical analysis of Gaucher disease allele c.1448T>C (L444P)

BackgroundGaucher disease is a potentially severe lysosomal storage disorder caused by mutations in the human glucocerebrosidase gene (GBA). We have developed a multiplexed genetic assay for eight diseases prevalent in the Ashkenazi population: Tay-Sachs, Gaucher type I, Niemann-Pick types A and B, mucolipidosis type IV, familial dysautonomia, Canavan, Bloom syndrome, and Fanconi anemia type C. This assay includes an allelic determination for GBA allele c.1448T>C (L444P). The goal of this study was to clinically evaluate this assay.MethodsBiotinylated, multiplex PCR products were directly hybridized to capture probes immobilized on fluorescently addressed microspheres. After incubation with streptavidin-conjugated fluorophore, the reactions were analyzed by Luminex IS100. Clinical evaluations were conducted using de-identified patient DNA samples.ResultsWe evaluated a multiplexed suspension array assay that includes wild-type and mutant genetic determinations for Gaucher disease allele c.1448T>C. Two percent of samples reported to be wild-type by conventional methods were observed to be c.1448T>C heterozygous using our assay. Sequence analysis suggested that this phenomenon was due to co-amplification of the functional gene and a paralogous pseudogene (ΨGBA) due to a polymorphism in the primer-binding site of the latter. Primers for the amplification of this allele were then repositioned to span an upstream deletion in the pseudogene, yielding a much longer amplicon. Although it is widely reported that long amplicons negatively impact amplification or detection efficiency in recently adopted multiplex techniques, this assay design functioned properly and resolved the occurrence of false heterozygosity.ConclusionAlthough previously available sequence information suggested GBA gene/pseudogene discrimination capabilities with a short amplified product, we identified common single-nucleotide polymorphisms in the pseudogene that required amplification of a larger region for effective discrimination.

Virus-Coated Layer-by-Layer Colloids as a Multiplex Suspension Array for the Detection and Quantification of Virus-Specific Antibodies

Suspension array technology has surpassed ELISA for automated, simultaneous detection and quantification of soluble biomarkers such as virus-specific antibodies. We describe assays in which antigens are attached to a lipid bilayer surrounding color-coded particles. We used layer-by-layer technology to establish a multiplex suspension array with distinguishable microbeads coated with authentic viral surfaces to catch and quantify virus-specific antibodies in a flow cytometric analysis. Antigenic surfaces were generated by chimeric and wild-type baculoviruses plus 2 different influenza A virus subtypes fused to a lipid bilayer surrounding distinctly colored particles. Specificity of binding of chosen antibodies and sera was detected by immunofluorescence. Results of multiplex analysis were compared with results of ELISA. Titrations of virus-specific antibodies in the multiplex suspension array demonstrated specific binding to the viral surface proteins. The multiplex suspension array gave positive results for up to log 5-diluted primary antibodies with an approximately 5- to 10-fold reduced dynamic range compared with the respective ELISA. The bead-based multiplex suspension array is customizable and easy to establish. By displaying native influenza A virus surfaces and recombinant HIV-1 epitopes, the new assay provides a tool for the detection of major viral infections in humans.

Inflammatory Cytokines in Lung Lavage Fluid Determined by Bead-Based and ELISA Cytokine Assays

RATIONALE: Multiplex bead-based suspension arrays enable simultaneously measurement of multiple analytes in a single sample compared to traditional biochemical methods that detect proteins individually. We sought to validate the multiplex suspension array system for analysis of cytokines in murine lung lavage samples.

Multiplex Analysis of Intracellular Signaling Pathways in Lymphoid Cells by Microbead Suspension Arrays

Phosphorylation analysis of signaling proteins is key for examining intracellular signaling pathways. Conventional biochemical approaches, e.g. immunoprecipitation, Western blot, and ELISA, have played a major role in elucidation of individual signaling events. However, these methods are laborious, time-consuming, and difficult to adapt for high throughput analysis. A multiplex approach to measure phosphorylation state of multiple signaling proteins simultaneously would significantly enhance the efficiency and scope of signaling pathway analysis for mechanistic studies and clinical application. This report describes a novel multiplex microbead suspension array approach to examine phosphoproteomic profiles in lymphoid cells. In the Jurkat T-cell leukemia line, the multiplex assay enabled targeted investigation of phosphorylation kinetics of signal transduction from receptor proximal events (tyrosine phosphoproteins CD3, Lck, Zap-70, and linker for T-cell activation) to cytosolic events (serine/threonine phosphoproteins Erk and Akt) to transcription factors (serine/threonine phosphorylated Rsk, cyclic AMP-response element-binding protein, and STAT3). To broaden the application of the multiplex analysis, signaling pathways were also studied in B-cell lymphoid tumor lines that included chronic lymphocytic leukemia lines. In these cell lines, multiplex suspension array enabled phosphoproteomic analysis of signaling cascade mediated by Syk, a homolog of Zap-70. Results obtained by multiplex analysis were confirmed by immunoprecipitation and Western blot methods. The examples of T-cell and B-cell signaling pathway analyses in this report demonstrate the utility of the multiplex suspension arrays to investigate phosphorylation dynamics and kinetics of several signaling proteins simultaneously in signal transduction pathways.

Fluorescent Nanometer Microspheres as a Reporter for Sensitive Detection of Simulants of Biological Threats Using Multiplexed Suspension Arrays

We succeeded in using 40 nm FRET (fluorescence resonance energy transfer) microspheres conjugated to antibodies as the fluorescent reporters to perform the multiplexing suspension array measurements on two simulants of biological threats, ricin (A chain) and a crude spore preparation of Bacillus globigii (Bg). The microspheres were impregnated with two types of fluorophores in equal number (approximately 140 fluorophores in total per microsphere) and displayed bright PE-like fluorescence via a fluorescence resonance energy transfer mechanism. Activated microspheres (aldehyde groups) were directly coupled to antibodies and used to form sandwich-type immunoassays in a suspension array. For the crude preparations of Bg, the assay sensitivity using antibody-conjugated microspheres is an order of magnitude higher than that using the conventional fluorescent reporter, R-phycoerythrin (PE). Using the microspheres, Bg at the concentration of 5 ng/mL can be easily detected. For ricin, the assay sensitivity was similar to that obtained using PE as the reporter, but washing the reaction mixtures resulted in the fluorescence signals that were 2-3 times higher compared to those using PE. Ricin at a concentration of 1 ng/mL can be readily identified. Importantly, the two simulants do not interfere with each other in the multiplexing experiments. The 40 nm FRET microspheres are a new sensitive alternative as fluorescent reporters for detection in suspension arrays.