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Thank you to all the attendees, speakers, and partners for making Planet xMAP USA 2007 a success. We look forward to seeing you at Planet xMAP USA 2008 in Boston, MA - March 3-5! (Adobe Reader required to view presentations)
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| Keynote Address - Monday, March 12 |
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Larry A. Sklar Ph.D., Regents Professor of Pathology and Distinguished Professor of Pharmacy
Director of Basic Research, Cancer Research and Treatment Center
University of New Mexico Health Sciences Center
Presentation: High Throughput Flow Cytometry of Multiplexed Targets for Drug Discovery
The high throughput (HT) flow cytometry platform HyperCyt is adept at both cell and particle-based assays and is compatible with both high content and multiplex analysis. Cell-based assays have initially been directed against G protein-coupled receptor (GPCR) targets where we have identified novel small molecule ligands for the novel GPR30 estrogen receptor. Other cell based screens have been conducted for the FPR family, the MDR family, bacterial virulence, and androgen pathways. We have developed general particle-based multiplexed approaches compatible with screening assemblies of soluble membrane receptors, protein domains, proteases, proteasome, kinases, nucleases, Bcl-2 and GTPase family proteins. We have used the soluble receptor approach to probe the mechanism of partial agonism of the b2-adrenergic receptor and the steps in signal transduction using flow cytometry-based kinetic analysis.
The NIH Roadmap Molecular Libraries Initiative (MLI) has given us the opportunity, through the New Mexico Molecular Libraries Screening Center (NMMLSCN, http://nmmlsc/) to implement HT flow cytometry for the international research community, and to integrate virtual and physical screening with medicinal chemistry to develop biological probes based on small molecule discovery. The WM Keck Foundation has given us the opportunity to evaluate small molecules as imaging agents through PET and SPECT with novel isotopes from Los Alamos National Labs.
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| Diagnostic Speakers - Tuesday, March 13 |
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Jim Mahony, Ph.D., Director of the McMaster University Regional Virology and Chlamydiology Laboratory and President of the Pan American Society for Clinical Virology
Presentation: Detection and Identification of 20 Respiratory Viruses Using Multiplex PCR and the Luminex xMAP System (presentation not available)
A new Respiratory Virus Panel test that detects and identifies 95% of all respiratory viruses in clinical specimens has recently been developed by TmBioscience Corporation. This test, called the ID-Tag™ RVP Test, uses a two-step multiplex RT-PCR (16 primer pairs), a multiplex Target Specific Primer Extension (TSPE) reaction (21 primers) and hybridization to a fluid microarray of 21 spectrofluorometrically-labeled microspheres containing complimentary virus-specific oligonucleotides that are sorted by the Luminex xMAP System and read on the Luminex 100 instrument. This assay is more sensitive than conventional tests and identifies 20 different respiratory viruses including emerging viruses such as SARS-CoV and Influenza H5N1. |
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Weimin Sun, Ph.D., Scientific Director, Molecular Genetics Department
Quest Diagnostics Nichols Institute
Presentation: Implementation of Multiplex Luminex-based Genotyping Assays in a High-Throughput Diagnostic Laboratory (presentation not available)
We will share our experience in implementing and performing multiplex DNA genotype detection assays based on different genotyping chemistries. Detection on the Luminex platform has enabled us to achieve a higher level of multiplexing when compared to fragment analysis detected on automated fluorescent DNA sequencers. The technology transformation has allowed our laboratory to obtain higher efficiency and improved workflow. Technical and other challenges during assay implementation will be discussed.
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Jim Versalovic, M.D., Ph.D., Director, Division of Molecular Pathology, Texas Children's Hospital Director, Microbiology Laboratories, Texas Children's Hospital Assistant Professor of Pathology, Molecular and Human Genetics, and Molecular Virology and Microbiology, Baylor College of Medicine
Presentation: Liquid Bead Arrays for the Diagnosis of Bacterial Sepsis
The diagnosis of sepsis currently depends on microbial isolation in semi-automated continuously monitoring blood culture systems. Positive blood cultures provide evidence of bacteremia that may be associated with sepsis and life-threatening systemic infections. Microscopic morphology of positive blood cultures provides the basis for DNA testing with liquid bead array-based pathogen panels. Selected panels comprised of beads linked with genus- and species-specific DNA oligomers may be used for rapid and accurate identification of likely bacterial pathogens. The ultimate goal is to develop global diagnostic systems that can detect potential sepsis cases using spectrally addressable liquid bead arrays. |
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Jean Amos Wilson, Ph.D., Scientific Director, Human Genetics
Focus Diagnostics, Inc
Presentation: The Migration of Cystic Fibrosis Testing to the Middle Market
Cystic fibrosis (CF) is an autosomal recessive, complex multisystem disease that affects the respiratory tract, pancreas, intestine, male genital tract, hepatobiliary system and exocrine sweat glands. Pulmonary disease is the major cause of morbidity and mortality in CF. Because carrier screening for any couple planning a pregnancy has been the standard of care since 2001 and since robust testing platforms are now available, CF testing has begun to migrate from esoteric reference laboratories to more routine testing environments. This presentation will review CF as a clinical and genetic entity and describe the key features of the testing market. |
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Christine Ginocchio, Ph.D., Director, Microbiology/Virology and Molecular Diagnostics
Long Island Jewish Health System Laboratories
Presentation: Multiplexed Molecular Testing for the Detection of Human Respiratory Viruses using the TM Bioscience ID-Tag™ RVP Assay
The timely identification of respiratory viral infections is of tremendous importance for diagnosis, infection control, epidemiology and surveillance studies. This study evaluated the ID-Tag™ RVP assay (Tm Bioscience, Toronto, Canada) for the detection of 19 strains and subtypes of respiratory viruses, including RSV (A, B), influenza A (H1, H3, H5), influenza B, parainfluenza (1, 2, 3, 4), adenovirus, coronavirus (229E, OC43, NL63, HKU1, SARS), human metapneumovirus (hMPV), and enterovirus/rhinovirus (E/R).
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Kodumudi Venkat Venkateswaran, Ph.D., Vice President, Research and Business Development
Radix BioSolutions
Presentation: Application of xMAP Technology for Personalized Medicine and Immune Monitoring of Cancer Vaccine Therapy
Autologous tumor cells grown in vitro have been of considerable value for active-specific immunotherapy and tumor immune response monitoring as they contain any and all relevant antigens for a particular patient. We designed a novel, easy to do and cost-effective method to monitor anti-tumor immune responses directly in patient samples during personalized melanoma vaccine therapy. In Phase I/II clinical trial, metastatic melanoma patients were treated with dendritic cells (DC) generated from each patient, co-cultured with autologous irradiated, IFNg-treated tumor cells (TC) and suspended in suitable medium. We developed a novel whole blood assay in conjunction with xMAP technology for immune monitoring of the vaccine-induced response. Serum and heparinized blood samples of vaccine recipients were collected before, during, and after therapy (week 0, 4 & 24). After in vitro stimulation of aliquots of each whole blood sample overnight with tumor antigens, the serum and secretory cytokine profiles were measured using Bio-Rad human 17-plex cytokine kit, Bioplex Suspension Array Reader and BioPlex Manager Software. Results of this immunoproteomics approach with whole blood testing show great promise to be applicable as a routine test for the personalized cancer immunotherapy. This cost-effective method is flexible, amenable to high-throughput and provides overall individualized evaluation of specific immune responses to autologous tumor cells by simultaneously measuring various secretory mediators.
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Mary McBride, Ph.D., Associate Division Leader, Medical Physics and Biophysics Division
Lawrence Livermore National Laboratory
Presentation: FluIDx: A Multiplexed Diagnostic Platform for Use at Point-of-Care
We have developed, tested, and piloted a diagnostic system – instrumentation and multiplexed assays - for the detection of influenza and other high-priority respiratory pathogens. The instrument, referred to as FluIDx, and the multiplexed assays were designed specifically for use at point-of-care (POC) to facilitate the diagnosis of patients who present in hospital emergency departments or clinics with respiratory symptoms. This system is capable of sample processing, performing multiplexed real-time PCR, data analysis, and reporting. All functions are completely automated. Here we describe the development, testing, and evaluation of the multiplexed assay panel, present an overview of the FluIDx system, and provide as assessment of the instrument’s utility as a POC diagnostic. |
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| Life Science Research Speakers - Wednesday, March 14 |
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Karen Ebsworth, Senior Associate Scientist
Amgen, Inc.
Presentation: Gene Expression Analysis by QuantigenePlex®: Validation and Applications (presentation not available)
Methods for the analysis of gene expression typically include quantitative RT-PCR (qPCR) or microarray technology, however these techniques have a number of limitations. QPCR has a low capacity to multiplex and requires amplification of the template. Microarray technology generates data on thousands of gene transcripts, many of which show no experimental regulation and are irrelevant to the field of study. The analysis of cytokine release, from in vitro activated cells or from animal models of inflammation, is often by immunoassay (e.g. ELISA) but this facilitates the analysis of only a single cytokine per assay. In addition to these limitations, all these methods can be time consuming and require large sample volumes.
In order to examine patterns of gene expression and cytokine release for both in vivo and in vitro models of inflammation, we have utilized xMAP® technology to multiplex both gene expression and corresponding cytokine release. Specifically, we have used Panomics’ Quantigene® Plex to analyze RNA samples and cell lysates, and Bio-Rad’s Bio-Plex system to analyze serum samples and cell culture supernatant for cytokine protein production. Thus we have been able to maximize data obtained from limited samples.
We will present data showing correlation of cytokine gene expression as measured by QuantiGene® Plex, with that measured by quantitative RT-PCR. Further, we will show that gene expression measured in the tissues of mice with acute inflammation correlates with cytokine serum levels measured by the Bio-Rad assay. We have also used xMAP® technology to assess compound activity in vitro through correlation of gene expression and cytokine release. Finally, through utilizing QuantiGene® Plex, we have evaluated the expression pattern of over 150 inflammation related genes in a mouse model of Rheumatoid Arthritis (Collagen-Induced Arthritis) and will show correlation between gene expression and clinical disease.
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John Ruzinski, B.S., Research Scientist, Pulmonary and Critical Care
Harborview Medical Center
Presentation: Identification of High and Low Cytokine Responders to Agonists in the Toll Like Receptor Pathway using xMAP Technology
Individual differences in responses to pathogens are important determinants of outcome in critically ill humans. Our current work is directed at understanding the inter-individual variation in responses to a large panel of Toll-Like Receptor (TLR) agonists in a large number of normal human volunteers (n > 1000). This talk will describe our use of xMAP technology to perform high throughput screening of cytokine production in culture supernatants of TLR agonist-stimulated whole blood samples . Comparisons to traditional ELISA will be made and descriptions of inter-assay and intra-assay variation will be discussed. |
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Paul Rhyne, Ph.D., Group Leader, Clinical Biomarker Development
Pharmaceutical Research Institute
Presentation: Comparative Analysis of 10 Multiplexed Cytokine Immunoassays Using Multiple Technology Platforms
Many technologies are commonly used to measure cytokine levels in human plasma including suspension bead arrays, antibody planer arrays, ELISA, and others. We developed 10 immunoassays
using commercially available antibodies for the detection of human cytokines. The performance of each of these assays both individually and multiplexed were assessed using different technologies. The
performance characteristics of these assays such as lower limit of quantitation, range, reproducibility, precision, and accuracy were calculated using a common set of cytokine standards and plasma
samples. A comparison of assay performance, ease of use, cost, and other assay characteristics will be discussed in this presentation. |
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Uday Veeramallu, Senior Director, Bioprocess Development
Biosite Discovery/Biosite Inc.
Presentation: Antibody Screening and Early Evaluation of Biomarkers Using xMAP Technology, Including Magnetic Microspheres
Recombinant antibody screening applications using xMAP technology are described. Mice immunizations and phage display vectors are used to generate recombinant antibody libraries for biomarkers. These libraries are cloned into plasmid vectors in E. coli. The recombinant antibodies are expressed, purified and conjugated to xMAP microspheres using custom conjugation chemistries. Examples illustrate the screening of antibody combinations and characterization of epitope diversity in the recombinant libraries. Derivative antibody pairs are used for developing sandwich immunoassays for the biomarkers. Illustrative examples, including the use of magnetic xMAP microspheres, further demonstrate the early evaluation of the utility of the biomarkers for diagnostic immunoassays. |
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Peter Zimmerman, Ph.D., Associate Professor, The Center for Global Health and Diseases
Case Western Reserve University
Presentation: A Multiplex Ligase Detection Reaction-Fluorescent Microsphere Assay (LDR-FMA) for Simultaneous Diagnosis of Single Nucleotide Polymorphisms Associated with Plasmodium falciparum Drug Resistance
Incomplete malaria control efforts have resulted in a worldwide increase in resistance to drugs used to treat the disease. A complex array of mutations underlying anti-malarial drug resistance complicates efficient monitoring of parasite populations and limits the success of malaria control efforts in endemic regions. To improve surveillance of Plasmodium falciparum (Pf) drug resistance in our Papua New Guinea based studies, we developed a multiplex ligase detection reaction/fluorescent microsphere based assay (LDR-FMA; Luminex® FlexMAP™ microsphere technology) that identifies SNPs associated with resistance to Fansidar and chloroquine in the Pf dhfr (9 alleles), dhps (10 alleles), and pfcrt (3 alleles) genes. |
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Leslie M. Shaw, Ph.D., Professor and Director, Clinical Toxicology Laboratory, Director, Xenobic Toxicokinetics Research Laboratory, Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center
Presentation: Characterization of the xMAP Technology and Performance of INNO-BIA ALZBIO3 for Quantification in CSF of the Alzheimer's Disease Biomarkers b-AMYLOID(1-42), TOTAL TAU and Phosphorylated TAU(181P) (presentation not available)
Three biomarkers measured in cerebrospinal fluid, b-amyloid (1-42), total Tau and phosphorylated Tau(181p), have been shown, using xMAP technology or individual ELISA methods, to have at least 85% diagnostic sensitivity and 80% specificity for detection of Alzheimer’s disease (AD), predicting mild cognitive impairment patients progressing to AD, and identifying elderly cognitively normal individuals likely to progress to memory impairment. Investigations are progressing for determination of the best therapies for delaying the development of AD in at-risk individuals and therefore there is increasing interest in validation of the tests for biomarkers of AD. In this presentation our experience at the University of Pennsylvania biomarker core laboratory of the Alzheimers Disease Neuroimaging Initiative (ADNI) in the validation of the xMAP technology using INNO-BIA ALZBIO3 bead-based immunoassay reagents and the Luminex platform will be described. |
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Rajeev Dhawan, Ph.D., Director, Diagnostic Reagents
Charles River Laboratories
Presentation: Development and Validation of Multiplexed Fluorometric Immunoassay® (MFIA®) for Serodiagnosis of Laboratory Rodents Infectious Diseases using Luminex xMAP® Technology
Serology is the principal diagnostic methodology by which laboratory animals are monitored for specific adventitious (i.e., accidental) infections that cause diseases or otherwise interfere with biomedical research. Using the Luminex xMAP technology, we at Charles River Laboratories have developed MFIA panels for serosurveillance of laboratory mice and rats. The mouse and rat panels comprise of 29 and 25 assays, respectively, for antibodies to a variety of infectious agents including viruses and other microbes. An important aspect of CGLP compliance is assay validation. Thus, a validation study was carried out to substantiate the suitability of the multiplex MFIA vs. singleplex ELISA and IFA for diagnosing, or detecting, infections of mice and rats with indigenous infectious agents. Rodent MFIA panels were tested using known-negative (KN) sera from specific-pathogen-free (SPF) Charles River Laboratories barrier-room colonies and microbial antibody known-positive (KP) sera from naturally and intentionally (i.e., experimentally) infected rodents. Reproducibility and ruggedness of MFIA was very high as tech to tech variation between different MFIA runs remained low. The limit of detection, selectivity and diagnostic performance of the MFIA were found to be comparable to or better than those obtained by ELISA and IFA. The diagnostic specificity of the mouse and rat MFIA were 100% and 99%, respectively. In comparison to ELISA, MFIA diagnostic sensitivity was ≥ 95% and the percentage agreement between MFIA, ELISA and IFA was ≥ 90%. |
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Ergang Shi, Ph.D., Senior Scientist
Regeneron Pharmaceuticals Inc.
Presentation: Monoclonal Antibody Classification Based on Epitope Binding Using Differential Antigen Disruption
The ability to process a large number of mAbs to an antigen based on their epitope diversity is essential for systematically screening desirable clones. This demand is challenging the traditional antigen screening assays as well as the traditional epitope mapping methodologies on speed, efficiency and throughput. To overcome these challenges, we’ve developed a novel antigen-structure-based screening approach, named as differential antigen disruption (DAD), to profile a large number of mAbs according to their epitope bindings. This method has been developed first by using sensor chips of Biacore and then by using xMAP® Carboxylated Microspheres of Luminex. The presentation will focus on the development and applications of the DAD technology for VelocImmune® human therapeutic antibody programs in Regeneron. |
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