Fig 1 - uploaded by James W Jacobson
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Identification of CFTR mutations by the Luminex LabMAP system. The bar graph (top) demonstrates the net median fluorescence for wild-type (normal) and mutant probes for each of the five targets in a normal DNA sample. Net median fluorescence values obtained for all 15 patient DNA samples are listed at the bottom. Shaded boxes indicate positive results.
Citations
... Currently, Luminex-based bioassays are being performed in a wide range of applications throughout the drug-discovery and diagnostics fields, as well as basic research. These applications include protein expression profiling (cancer and metabolic markers [17,18], cytokines [11,[19][20][21], growth factors [22], endocrine [23], matrix metalloproteinases [24], transcription factors/nuclear receptors [25]); genomic research (gene expression profiling, genotyping, and microRNA array) [26,27]; genetic disease (cystic fibrosis and cytochrome p450) [28,29]; and immunodiagnostics (allergy and vaccine testing, autoimmune disease, HLA testing, infectious disease, and newborn screening) [14,[30][31][32]. ...
Immunoassay is one of the most commonly used biomedical techniques to detect the expression of an antibody or an antigen in a test sample. Enzyme-linked immunosorbent assay (ELISA) has been used for a variety of applications including diagnostic tools and quality controls. However, one of the main limitations of ELISA is its lack of multiplexing ability, so ELISA may not be an efficient diagnostic tool when a measurement of multiple determinants is needed for samples with limited quantity such as blood or biological samples from newborns or babies. Although similar to ELISA in assay measurement, multiplex platforms such as bead-based Luminex and multi-array-based MSD (Meso Scale Discovery) are widely used to measure multiple biomarkers from a single analysis. Luminex is a xMAP-based technology that combines several different technologies to provide an efficient and accurate measurement of multiple analytes from a single sample. The multiplexing can be achieved because up to 100 distinct Luminex color-coded microsphere bead sets can be coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes from a sample. Using Multi-array and electrochemiluminescence technologies, the MSD platform provides the multiplex capability with similar consistence as observed in ELISA. Various biological samples that can be analyzed by both Luminex and MSD systems include serum, plasma, tissue and cell lysate, saliva, sputum, and bronchoalveolar Lavage (BAL). The most common Luminex and MSD-based assays are to detect a combined set of cytokines to provide a measurement of cytokine expression profiling for a diagnostic purpose.
... Using the same strategy, the addition of four supplementary layers will theoretically permit the analysis of 500 different analytes in the future. Different analytes can be quantified since different capture molecules can be conjugated on the beads' surface allowing for multiple uses in many clinical laboratory areas: genotyping, cytokine and thyroid level measurements, cysticfibrosis screening, genetic human lymphocyte antigen (HLA) typing, kinase testing, and allergy testing [1][2][3][4][5][6][7][8][9][10][11][12][13]. ...
BACs-on-Beads™ (BoBs™) is a new emerging technology, a modification of comparative genomic hybridization that can be used to detect DNA copy number gains and losses. Here, we describe the application of two different types of BoBs™ assays: (1) Prenatal BoBs (CE-IVD) to detect the most frequent syndromes associated with chromosome microdeletions, as well as the trisomy 13, 18 and 21, and (2) KaryoLite BoBs (RUO) which can detect aneuploidy in all chromosomes by quantifying proximal and terminal regions of each chromosomal arm. The interpretation of the results by BoBsoft™ software is also described. Although BoBs™ may not have the breadth and scope to replace chromosomal microarrays (array comparative genomic hybridization and single nucleotide polymorphism array) in the prenatal setting, particularly when a fetal anomaly has been detected, it is a well suited alternative for FISH or QF-PCR because BoBs™ is comparable, if not superior in terms of cost, turnaround time (TAT) and throughput and accuracy. BoBs™ also has the ability to detect significant fetal mosaicism (≥30 % with Prenatal BoBs and ≥50 % with KaryoLite BoBs). However, perhaps the greatest strength of this new technology is the fact that unlike FISH or QF-PCR, it has the ability to detect common microdeletion syndromes or additional aneuploidies, both of which may be easily missed despite excellent prenatal sonography. Thus, when BoBs™ is applied in the correct clinical setting and run and analyzed in appropriate laboratories this technique can improve and augment best practices with a personalization of prenatal care.
... Exonuclease I and shrimp alkaline phosphatase (EXO-SAP) reaction was performed in a 25-ml volume containing: 1 Â SAP buffer, 1 U shrimp alkaline phosphatase (inactivating any remaining nucleotides) (TaKaRa), 10 U exonuclease I (degrading any remaining PCR primers) (New England Biolabs, Ipswich, USA) and 7.5 ml PCR product. Samples According to Luminex's one-step carbodiimide coupling procedure [17], probes were coupled to carboxylated microspheres. Each coated bead population was suspended and stored in TE buffer. ...
... Currently, Luminex-based bioassays are being performed in a wide range of applications throughout the drug-discovery and diagnostics fields, as well as basic research. These applications include protein expression profiling (cancer and metabolic markers [18,19], cytokines [15,[20][21][22], growth factors [23], endocrine [24], matrix metalloproteinases [25], transcription factors/ nuclear receptors [26]); genomic research (gene expression profiling, genotyping, and microRNA array) [27,28]; genetic disease (cystic fibrosis and cytochrome p450) [29,30]; and immunodiagnostics (allergy and vaccine testing, autoimmune disease, HLA testing, infectious disease, and newborn screening) [8,[31][32][33] ...
Immunoassays are one of the most commonly used biomedical techniques to detect the expression of an antibody or an antigen in a test sample. Enzyme-linked immunosorbent assay (ELISA) has been used for various applications including diagnostic tools and quality controls. However, one of the main limitations of ELISA is its lack of multiplexing ability, so ELISA may not be an efficient diagnostic tool when a measurement of multiple determinants is needed for samples with limited quantity such as blood or biological samples from newborns or babies. Although similar to ELISA in assay measurement, Luminex is an xMAP-based technology that combines several different technologies to provide an efficient and accurate measurement of multiple analytes from a single sample. The multiplexing can be achieved because up to 100 distinct Luminex color-coded microsphere bead sets can be coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes from a sample. Various biological samples can be analyzed by a Luminex system include serum, plasma, tissue and cell lysate, saliva, sputum, and bronchoalveolar lavage (BAL). The most common Luminex-based assays are used to detect a combined set of cytokines to provide a measurement of cytokine expression profiling for a diagnostic purpose.
... Many microfluidic-based platforms have been developed for bioanalysis, including cell-based assays (Xu et al., 2010Xu et al., , 2013 Yue et al., 2013), nucleic acid analysis (Huang et al., 2006), protein engineering (Joensson and Andersson-Svahn, 2011), mutation detection (Han et al., 2011) and point-of-care testing (Holland and Kiechle, 2005; Tudos et al., 2001). Recently, the combinations of bead-based assays with microfluidic systems have attracted increasing attention due to the high-throughput of the microfluidic systems and the multiplexing capability of barcoded microbeads (Dendukuri et al., 2006;Dunbar and Jacobson, 2000; Nicewarner-Pena et al., 2001; Pregibon et al., 2007). Various bead-based bioassays integrated with microfluidic systems have been reported, including discrimination of single-nucleotide mismatches (Ali et al., 2003; Ng et al., 2007), genotyping of hepatitis B and HPV viruses (Zhang et al., 2010; Zhang et al., 2011) and influenza A virus (Lien et al., 2011). ...
In this study, a microfluidic platform was developed to generate single layer, linear array of microbeads for multiplexed high-throughput analysis of biomolecules. The microfluidic device is comprised of eight microbead-trapping units, where microbeads were immobilized in a linear array format by the exertion of a negative pressure in the control channel connected to each sieving microstructure. Multiplexed assays were achieved by using a mixture of different spectrally-encoded microbeads functionalized with specific probes, followed by on-chip reaction and detection. The microfluidic-based microbeads array platform was employed for multiplexed analysis of DNA and proteins, as demonstrated by the simultaneous discrimination of four HPV genotypes and the parallel detection of six different proteins. Compared with the off-chip protocols, the on-chip analysis exhibited better reaction efficiency, higher sensitivity and wider linear detection range. Visual inspection and identification of functionalized microbeads were facilitated by the single layer arrangement of microbeads so that accurate data acquisition can be performed during the detection process.
... 15,16 Likewise, solution hybridization of multiple PCR products on addressable microspheres, followed by flow cytometry fluorescence analysis, is broadly used in clinical diagnostic applications for bacterial, viral, genetic, or oncology targets. [17][18][19][20][21] Regardless of the technology platform, molecular methods currently in clinical use have been optimized to be compatible with anatomic pathology specimens. Because of the breadth of specimen types and preanalytic parameters commonly encountered in the clinical setting, the quantity or quality of nucleic acids recovered from these specimens can vary greatly. ...
Context:
Current clinicopathologic assessment of malignant neoplastic diseases entails the analysis of specific genetic alterations that provide diagnostic, prognostic, or therapy-determining information.
Objective:
To develop and validate a robust molecular method to detect clinically relevant mutations in various tissue types and anatomic pathology specimens.
Design:
Genes of interest were amplified by multiplex polymerase chain reaction and sequence variants identified by liquid bead array cytometry. The BRAF assay was fully characterized by using plasmids and genomic DNA extracted from cell lines, metastatic colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissues, and thyroid nodule fine-needle aspirates.
Results:
Qualitative multiplex assays for 22 different mutations in the BRAF, HRAS, KRAS, NRAS, or EGFR genes were established. The high signal-to-noise ratio of the technology enabled reproducible detection of BRAF c.1799T>A (p.V600E) at 0.5% mutant allele in 20 ng of genomic DNA. Precision studies with multiple operators and instruments showed very high repeatability and reproducibility with 100% (98.7%-100%) qualitative agreement among 292 individual measures in 38 runs. Evaluation of 1549 representative pathologic specimens in 2 laboratories relative to independent reference methods resulted in 99.0% (97.6%-99.6%) agreement for colorectal FFPE tissues (n = 416) and 98.9% (98.2%-99.4%) for thyroid fine-needle aspiration specimens (n = 1133) with an overall diagnostic odds ratio of 10 856 (2451-48 078).
Conclusions:
The multiplex assay system is a sensitive and reliable method to detect BRAF c.1799T>A mutation in colorectal and thyroid lesions. This optimized technology platform is suitable for the rapid analysis of clinically actionable genetic alterations in cytologic and histologic specimens.
... Liquid bead array hybridization followed by flow cytometry detection may be better suited to the requirements of the diagnostic laboratory. This format has been generally applied to clinical analytes, including serologic, genetic, bacterial and viral targets using the CEmarked and FDA-cleared Luminex ® 100 or 200 Systems (7)(8)(9)(10). We took advantage of the 96-well plate format and automation capabilities of this platform to develop a rapid multiplex assay for the qualitative detection of twelve fusion transcripts resulting from seven chromosomal abnormalities commonly found in chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), or acute myeloid leukemia (AML). ...
... Hybridization was performed according to the procedure described by Karpasitou et al. [4]. Each probe was covalently coupled to a different set of fluorescent polystyrene carboxylated microspheres, purchased internally labeled and ready to couple from the Luminex Corporation, by a carbodiimide method in separate reactions [8,9]. ...
BACKGROUND: In a previous publication we described a method for Jk(a)/Jk(b), Fy(a)/Fy(b), S/s, K/k, Kp(a)/Kp(b), Js(a)/Js(b), Co(a)/Co(b), and Lu(a)/Lu(b) genotyping based on a microsphere suspension array. Here, an improved version of the assay is presented. METHODS: TWO MULTIPLEX POLYMERASE CHAIN REACTIONS (PCR) WERE DEVELOPED: one for amplification of samples routinely tested and the other for those systems that are tested less frequently. Each biotinylated PCR product is hybridized in a single multiplex assay. A total of 2,020 samples were analyzed, and the genotypes were compared to the blood group phenotypes. RESULTS: There have been no discrepancies with the serology results other than null and/or weak phenotypes. CONCLUSION: In its present form, the method presented here has the capacity to genotype hundreds of a samples in few hours with a high concordance rate with serology.
... Immunoassays, western blot, immunohistochemical staining and real-time PCR have been extensively applied in this context. New multiplex immunoassay technologies such as Luminex or AlphaScreen (reviewed in Vignali (2000) and Taouji et al. (2009)), permitting measurement of multiple analytes in a single ELISA format, offer an ideal complement to proteomics studies requiring validation of multiple candidates (Dunbar & Jacobson 2000). Commercially available kits allow the simultaneous analysis of up to 42 different cytokines in a single assay (Richens et al. 2010). ...
Over the past decade, high-throughput proteomics technologies have evolved considerably and have become increasingly more commonly applied to the investigation of female reproductive diseases. Proteomic approaches facilitate the identification of new disease biomarkers by comparing the abundance of hundreds of proteins simultaneously to find those specific to a particular clinical condition. Some of the best studied areas of female reproductive biology applying proteomics include gynaecological cancers, endometriosis and endometrial infertility. This review will discuss the progress that has been made in these areas and will highlight some of the emerging technologies that promise to contribute to better understanding of the female reproductive disease.
... 38,39,40 For example, the Luminex xMAP system (commercial product by Luminex Corporation) uses 100 types of microspheres with 100 different colors, which can be identified by two laser beams, in genetic human lymphocyte antigen (HLA) typing and allergy testing. [41][42][43][44][45] Barcoded nano/microparticles provide a new and promising opportunity for disease diagnosis, combinatorial library, drug screening and drug discovery. The next section will introduce recent developments in barcoded nano/microparticles, also called microcarriers, and their various applications. ...
... Luminex color coded beads system has shown great performance in many applications, throughout the drug-discovery and diagnostics fields, as well as in basic research. [41][42][43][44][45] As stated in section 1. Quantum Dot Corporation has developed QD encoded microspheres with nine different spectral codes (two colors at three intensities) to identify ten different single nucleotide polymorphisms (SNPs). ...
