Using Luminex Corporation’s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.
Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles
1Department of Chemistry, Tufts University, 2Department of Analytical Chemistry, Complutense University (Spain), 3Department of Electrical and Computer Engineering, Tufts University
We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 μm polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.
Published October 10, 2013. Keywords: Chemistry, protein sensing, microarray, multiplexed fluorescent quantification, fiber-optic biosensor, microsphere-based immunoassays, saliva analysis, microsphere encoding
A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay
1Department of Cell and Molecular Physiology, Loyola University Chicago
Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.
Published August 8, 2013. Keywords: Molecular Biology, Cellular Biology, Biochemistry, Genetics, Biomedical Engineering, Medicine, Cardiology, Heart Diseases, Myocardial Ischemia, Myocardial Infarction, Cardiovascular Diseases, cardiovascular disease, immunoassay, cardiac myosin binding protein-C, cardiac troponin I, creatine kinase MB, electrochemiluminescence, multiplex biomarkers, ELISA, assay
An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.
Published July 6, 2012. Keywords: Molecular Biology, Luminex, xMAP, Multiplex, MAGPIX, MagPlex Low Concentration Microspheres, xMAP Antibody Coupling Kit, ELISA, Immunoassay, Antibody Screening, Optimization, Conversion
Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development
1Department of Internal Medicine, Division of Hematology and Oncology, University of California, Davis, 2Comparative Pathology Laboratory, UC Davis School of Veterinary Medicine, University of California, Davis, 3Merck Serono Research, Merck KGaA, Darmstadt, Germany
An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.
Published October 30, 2013. Keywords: Medicine, Urinary Bladder, Animals, Genetically Modified, Cancer Vaccines, Immunotherapy, Animal Experimentation, Models, Neoplasms Bladder Cancer, C57BL/6 Mouse, MUC1, Immunotherapy, Preclinical Model
Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples
1Institute for Hepatitis and Virus Research, 2Department of Microbiology and Immunology, Thomas Jefferson University, 3Drexel University College of Medicine, 4Van Andel Research Institute, 5Institute for Hepatitis and Virus Research, Serome Biosciences Inc.
In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.
Published May 4, 2012. Keywords: Molecular Biology, Glycoproteins, glycan-binding protein, specific protein glycosylation, multiplexed high-throughput glycan blocked antibody microarray
Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays
1Department of Biology, The University of Mississippi
Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.
Published October 1, 2013. Keywords: Environmental Sciences, Environmental Monitoring, Ecological and Environmental Processes, Environmental Microbiology, Ecology, extracellular enzymes, freshwater microbiology, soil microbiology, microbial activity, enzyme activity
To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation.
Published November 15, 2013. Keywords: Environmental Sciences, Ecological and Environmental Phenomena, Environment, Biochemistry, Environmental Microbiology, Soil Microbiology, Ecology, Eukaryota, Archaea, Bacteria, Soil extracellular enzyme activities (EEAs), fluorometric enzyme assays, substrate degradation, 4-methylumbelliferone (MUB), 7-amino-4-methylcoumarin (MUC), enzyme temperature kinetics, soil
Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing
1Department of Pathology, Memorial Sloan-Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center
We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.
Published October 18, 2013. Keywords: Molecular Biology, Molecular Diagnostic Techniques, High-Throughput Nucleotide Sequencing, Genetics, Neoplasms, Diagnosis, Massively parallel sequencing, targeted exon sequencing, hybridization capture, cancer, FFPE, DNA mutations
We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.