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.
1Center for Stroke Research Berlin (CSB), Charité - Universitätsmedizin Berlin, 2Klinik und Hochschulambulanz für Neurologie, Charité - Universitätsmedizin Berlin, 3Medical School of the Universität Hamburg, Universitätsklinikum Hamburg - Eppendorf, 4Berliner Feuerwehr, 5STEMO-Consortium
Identification of suspected stroke in the dispatch center of the Berlin Fire Brigade prompts the deployment of a CT-equipped ambulance. If ischemic stroke is confirmed and contraindications are excluded prehospital thrombolysis is applied.
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.
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.
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.
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.
1Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation
A protocol is described that uses Illumina's Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.
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.