Enzyme-Linked Immunosorbant Assay (ELISA)
Total Page:16
File Type:pdf, Size:1020Kb
Load more
Recommended publications
-
"Epitope Mapping: B-Cell Epitopes". In: Encyclopedia of Life Sciences
Epitope Mapping: B-cell Advanced article Epitopes Article Contents . Introduction GE Morris, Wolfson Centre for Inherited Neuromuscular Disease RJAH Orthopaedic Hospital, . What Is a B-cell Epitope? . Epitope Mapping Methods Oswestry, UK and Keele University, Keele, Staffordshire, UK . Applications Immunoglobulin molecules are folded to present a surface structure complementary to doi: 10.1002/9780470015902.a0002624.pub2 a surface feature on the antigen – the epitope is this feature of the antigen. Epitope mapping is the process of locating the antibody-binding site on the antigen, although the term is also applied more broadly to receptor–ligand interactions unrelated to the immune system. Introduction formed of highly convoluted peptide chains, so that resi- dues that lie close together on the protein surface are often Immunoglobulin molecules are folded in a way that as- far apart in the amino acid sequence (Barlow et al., 1986). sembles sequences from the variable regions of both the Consequently, most epitopes on native, globular proteins heavy and light chains into a surface feature (comprised of are conformation-dependent and they disappear if the up to six complementarity-determining regions (CDRs)) protein is denatured or fragmented. Sometimes, by acci- that is complementary in shape to a surface structure on the dent or design, antibodies are produced against linear antigen. These two surface features, the ‘paratope’ on the (sequential) epitopes that survive denaturation, though antibody and the ‘epitope’ on the antigen, may have a cer- such antibodies usually fail to recognize the native protein. tain amount of flexibility to allow an ‘induced fit’ between The simplest way to find out whether an epitope is confor- them. -
Ten Years of Lateral Flow Immunoassay Technique Applications: Trends, Challenges and Future Perspectives
sensors Review Ten Years of Lateral Flow Immunoassay Technique Applications: Trends, Challenges and Future Perspectives Fabio Di Nardo * , Matteo Chiarello , Simone Cavalera , Claudio Baggiani and Laura Anfossi Department of Chemistry, University of Torino, 10125 Torino, Italy; [email protected] (M.C.); [email protected] (S.C.); [email protected] (C.B.); [email protected] (L.A.) * Correspondence: [email protected] Abstract: The Lateral Flow Immunoassay (LFIA) is by far one of the most successful analytical platforms to perform the on-site detection of target substances. LFIA can be considered as a sort of lab-in-a-hand and, together with other point-of-need tests, has represented a paradigm shift from sample-to-lab to lab-to-sample aiming to improve decision making and turnaround time. The features of LFIAs made them a very attractive tool in clinical diagnostic where they can improve patient care by enabling more prompt diagnosis and treatment decisions. The rapidity, simplicity, relative cost-effectiveness, and the possibility to be used by nonskilled personnel contributed to the wide acceptance of LFIAs. As a consequence, from the detection of molecules, organisms, and (bio)markers for clinical purposes, the LFIA application has been rapidly extended to other fields, including food and feed safety, veterinary medicine, environmental control, and many others. This review aims to provide readers with a 10-years overview of applications, outlining the trends for the main application fields and the relative compounded annual growth rates. Moreover, future perspectives and challenges are discussed. Citation: Di Nardo, F.; Chiarello, M.; Cavalera, S.; Baggiani, C.; Anfossi, L. -
Comparison of Immunohistochemistry with Immunoassay (ELISA
British Journal of Cancer (1999) 79(9/10), 1534–1541 © 1999 Cancer Research Campaign Article no. bjoc.1998.0245 Comparison of immunohistochemistry with immunoassay (ELISA) for the detection of components of the plasminogen activation system in human tumour tissue CM Ferrier1, HH de Witte2, H Straatman3, DH van Tienoven2, WL van Geloof1, FJR Rietveld1, CGJ Sweep2, DJ Ruiter1 and GNP van Muijen1 Departments of 1Pathology, 2Chemical Endocrinology and 3Epidemiology, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands Summary Enzyme-linked immunosorbent assay (ELISA) methods and immunohistochemistry (IHC) are techniques that provide information on protein expression in tissue samples. Both methods have been used to investigate the impact of the plasminogen activation (PA) system in cancer. In the present paper we first compared the expression levels of uPA, tPA, PAI-1 and uPAR in a compound group consisting of 33 cancer lesions of various origin (breast, lung, colon, cervix and melanoma) as quantitated by ELISA and semi-quantitated by IHC. Secondly, the same kind of comparison was performed on a group of 23 melanoma lesions and a group of 28 breast carcinoma lesions. The two techniques were applied to adjacent parts of the same frozen tissue sample, enabling the comparison of results obtained on material of almost identical composition. Spearman correlation coefficients between IHC results and ELISA results for uPA, tPA, PAI-1 and uPAR varied between 0.41 and 0.78, and were higher for the compound group and the breast cancer group than for the melanoma group. Although a higher IHC score category was always associated with an increased median ELISA value, there was an overlap of ELISA values from different scoring classes. -
Immunoassay - Elisa
IMMUNOASSAY - ELISA PHUBETH YA-UMPHAN National Institute of Health, Department of Medical Sciences 0bjective After this presentation, participants will be able to Explain how an ELISA test determines if a person has certain antigens or antibodies . Explain the process of conducting an ELISA test. Explain interactions that take place at the molecular level (inside the microtiter well) during an ELISA test. Outline - Principal of immunoassay - Classification of immunoassay Type of ELISA - ELISA ELISA Reagents General - Applications Principal of ELISA ELISA workflow What is immunoassay? Immunoassays are bioanalytical methods that use the specificity of an antigen-antibody reaction to detect and quantify target molecules in biological samples. Specific antigen-antibody recognition Principal of immunoassay • Immunoassays rely on the inherent ability of an antibody to bind to the specific structure of a molecule. • In addition to the binding of an antibody to its antigen, the other key feature of all immunoassays is a means to produce a measurable signal in response to the binding. Classification of Immunoassays Immunoassays can be classified in various ways. Unlabeled Labeled Competitive Homogeneous Noncompetitive Competitive Heterogeneous Noncompetitive https://www.sciencedirect.com/science/article/pii/S0075753508705618 Classification of Immunoassays • Unlabeled - Immunoprecipitation • Labeled Precipitation of large cross-linked Ag-Ab complexes can be visible to the naked eyes. - Fluoroimmnoassay (FIA) - Radioimmunoassay (RIA) - Enzyme Immunoassays (EIA) - Chemiluminescenceimmunoassay(CLIA) - Colloidal Gold Immunochromatographic Assay (ICA) https://www.creative-diagnostics.com/Immunoassay.htm Classification of Immunoassays • Homogeneous immunoassays Immunoassays that do not require separation of the bound Ag-Ab complex. (Does not require wash steps to separate reactants.) Example: Home pregnancy test. • Heterogeneous immunoassays Immunoassays that require separation of the bound Ag-Ab complex. -
Clinical Pharmacology 1: Phase 1 Studies and Early Drug Development
Clinical Pharmacology 1: Phase 1 Studies and Early Drug Development Gerlie Gieser, Ph.D. Office of Clinical Pharmacology, Div. IV Objectives • Outline the Phase 1 studies conducted to characterize the Clinical Pharmacology of a drug; describe important design elements of and the information gained from these studies. • List the Clinical Pharmacology characteristics of an Ideal Drug • Describe how the Clinical Pharmacology information from Phase 1 can help design Phase 2/3 trials • Discuss the timing of Clinical Pharmacology studies during drug development, and provide examples of how the information generated could impact the overall clinical development plan and product labeling. Phase 1 of Drug Development CLINICAL DEVELOPMENT RESEARCH PRE POST AND CLINICAL APPROVAL 1 DISCOVERY DEVELOPMENT 2 3 PHASE e e e s s s a a a h h h P P P Clinical Pharmacology Studies Initial IND (first in human) NDA/BLA SUBMISSION Phase 1 – studies designed mainly to investigate the safety/tolerability (if possible, identify MTD), pharmacokinetics and pharmacodynamics of an investigational drug in humans Clinical Pharmacology • Study of the Pharmacokinetics (PK) and Pharmacodynamics (PD) of the drug in humans – PK: what the body does to the drug (Absorption, Distribution, Metabolism, Excretion) – PD: what the drug does to the body • PK and PD profiles of the drug are influenced by physicochemical properties of the drug, product/formulation, administration route, patient’s intrinsic and extrinsic factors (e.g., organ dysfunction, diseases, concomitant medications, -
Development of a Prototype Lateral Flow Immunoassay for Rapid Detection of Staphylococcal Protein a in Positive Blood Culture Samples
diagnostics Article Development of a Prototype Lateral Flow Immunoassay for Rapid Detection of Staphylococcal Protein A in Positive Blood Culture Samples Arpasiri Srisrattakarn 1 , Patcharaporn Tippayawat 1, Aroonwadee Chanawong 1, Ratree Tavichakorntrakool 1, Jureerut Daduang 1, Lumyai Wonglakorn 2 and Aroonlug Lulitanond 1,* 1 Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; [email protected] (A.S.); [email protected] (P.T.); [email protected] (A.C.); [email protected] (R.T.); [email protected] (J.D.) 2 Clinical Microbiology Unit, Srinagarind Hospital, Khon Kaen University, Khon Kaen 40002, Thailand; [email protected] * Correspondence: [email protected]; Tel.: +66-(0)-4320-2086 Received: 11 August 2020; Accepted: 21 September 2020; Published: 7 October 2020 Abstract: Bloodstream infection (BSI) is a major cause of mortality in hospitalized patients worldwide. Staphylococcus aureus is one of the most common pathogens found in BSI. The conventional workflow is time consuming. Therefore, we developed a lateral flow immunoassay (LFIA) for rapid detection of S. aureus-protein A in positive blood culture samples. A total of 90 clinical isolates including 58 S. aureus and 32 non-S. aureus were spiked in simulated blood samples. The antigens were extracted by a simple boiling method and diluted before being tested using the developed LFIA strips. The results were readable by naked eye within 15 min. The sensitivity of the developed LFIA was 87.9% (51/58) and the specificity was 93.8% (30/32). When bacterial colonies were used in the test, the LFIA provided higher sensitivity and specificity (94.8% and 100%, respectively). -
Challenges Associated with the Development and Validation of Flow Cytometry Assays
Challenges associated with the development and validation of flow cytometry assays Carolyne Dumont, Eliane Moisan, Martin Poirier, Marie-Hélène Côté, and Marie-Soleil Piché 1 INTRODUCTION 2 Case Study 1 3 Case Study 2 Development of a PD marker by flow cytometry to assess the efficacy of Development of a flow cytometry assay for the measurement of basophil Flow cytometry is a technology allowing multi-parametric analysis of thousands of particles per second and helps to a chemokine neutralizing antibody activation in the context of a Phase III clinical study adequately identify or functionally characterize complex cell populations of interest. It is often used in basic research, Assay Design: The assay was required for the evaluation of pharmacodynamics (inhibition an agonist’s granulocytes activation activity) in Assay Design: Human whole blood samples were spiked with the different controls (or compounds in the clinical study) and further discovery, preclinical and clinical trials. With the increasing proportion of biologics in the pipeline, flow cytometry has proven preclinical studies. Non-Human Primate (NHP) or rat blood from treated animals was incubated at 3 conditions and granulocyte activation stained with an anti-CCR3 and anti-CD63 antibody. The validations stimulation conditions tested included a negative control (PBS) and itself to be an indispensable tool to assess safety, receptor occupancy (RO) or pharmacodynamics (PD). was measured as CD11b expression by flow cytometry (MFI). The conditions tested included a negative control (PBS), a positive control two positive controls (anti-FcεRI and fMLP). Basophils were identified as CCR3+ and upon activation, CD63 became externalized and (fMLP) and the test condition (agonist). -
Laboratory Techniques Used for Immunological Laboratory Methods
Laboratory techniques used for Immunological laboratory methods Dr. Tatiana Jones, MD, PhD NCC How to Make Serial Dilutions? Interpretation can be made differently depending on the nature of test. For example, if we need to figure out in what sample the concentration of the antibody or antigen is higher, we will go by TITER, which is the lowest serial dilution (let’s say that it is 1:32 in the picture on the left) that gives us positive result. This mean that even diluted 32 times sample is still capable of reacting. The other scenario when we are interpreting quantitative assays, such as ELISA. In this case we need to match results of our samples to known concentrations of STANDARD and MULTIPLY be our dilution factor. What is Antibody Titer? An antibody titer is a measurement of how much antibody an organism has produced that recognizes a particular antigen. Titer is expressed as the inverse of the greatest dilution that still gives a positive result. ELISA is a common means of determining antibody titers. How to Determine Antibody Titer? Where we can use Indirect Coombs test detects the presence of anti-Rh antibodies in blood serum. A patient might be reported to have an "indirect Antibody Titer? Coombs titer" of 16. This means that the patient's serum gives a positive indirect Coombs test at any dilution down to 1/16 (1 part serum to 15 parts diluent). At greater dilutions the indirect Coombs test is negative. If a few weeks later the same patient had an indirect Coombs titer of 32 (1/32 dilution which is 1 part serum to 31 parts diluent), this would mean that more anti-Rh antibody was made, since it took a greater dilution to eradicate the positive test. -
The Immunoassay Guide to Successful Mass Spectrometry
The Immunoassay Guide to Successful Mass Spectrometry Orr Sharpe Robinson Lab SUMS User Meeting October 29, 2013 What is it ? Hey! Look at that! Something is reacting in here! I just wish I knew what it is! anti-phospho-Tyrosine Maybe we should mass spec it! Coffey GP et.al. 2009 JCS 22(3137-44) True or false 1. A big western blot band means I have a LOT of protein 2. One band = 1 protein Big band on Western blot Bands are affected mainly by: Antibody affinity to the antigen Number of available epitopes Remember: After the Ag-Ab interaction, you are amplifying the signal by using an enzyme linked to a secondary antibody. How many proteins are in a band? Human genome: 20,000 genes=100,000 proteins There are about 5000 different proteins, not including PTMs, in a given cell at a single time point. Huge dynamic range 2D-PAGE: about 1000 spots are visible. 1D-PAGE: about 60 -100 bands are visible - So, how many proteins are in my band? Separation is the key! Can you IP your protein of interest? Can you find other way to help with the separation? -Organelle enrichment -PTMs enrichment -Size enrichment Have you optimized your running conditions? Choose the right gel and the right running conditions! Immunoprecipitation, in theory Step 1: Create a complex between a desired protein (Antigen) and an Antibody Step 2: Pull down the complex and remove the unbound proteins Step 3: Elute your antigen and analyze Immunoprecipitation, in real life Flow through Wash Elution M 170kDa 130kDa 100kDa 70kDa 55kDa 40kDa 35kDa 25kDa Lung tissue lysate, IP with patient sera , Coomassie stain Rabinovitch and Robinson labs, unpublished data Optimizing immunoprecipitation You need: A good antibody that can IP The right beads: i. -
An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate
Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-1981 An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate Allen H. Smith Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biochemistry Commons Recommended Citation Smith, Allen H., "An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate" (1981). All Graduate Theses and Dissertations. 5295. https://digitalcommons.usu.edu/etd/5295 This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. AN ENZYME- LINKED ThltviUNOSORBE!'IT ASSAY (ELISA) FOR PANTOTHENATE by Allen H. Smith A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Biochemistry UTAH STATE UNIVERSITY Logan, Utah 1981 ii ACKNOWLEDGEMENTS To Dr. R. G. Hansen, to Dr. B. W. Wyse, to Carl Wittwer, to Jack Brown, to Jan Pearson, to Nedra Christensen, to all those who have made this experience one of tremendous growth, I express my thanks. I express appreciation to the United States Department of Agriculture, under Grant #5901-0410-9-0288-0 with Utah State University, for financial support. Finally, I express thanks to my parents, who have come to realize that graduate school is also a part of life. Allen H. Smith "iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF TABLES . vi LIST OF FIGURES. vii · ABSTRACT .. ix INTRODUCTION 1 REVIEW OF LITERATURE 4 Pantothenate Assays . -
Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes
Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes Paula Denney Eason, Renée C. Benson, Jenny T. Ly, Rachel A. Saxer, James L. Wilbur, George B. Sigal, Eli N. Glezer, Hans A. Biebuyck, Jacob N. Wohlstadter, and Robert M. Umek TM TM A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes Abstract This poster presents a robust, receptor-ligand binding assay based upon a novel assay platform developed by Meso Scale DiscoveryTM (MSDTM). MSD’s platform combines array technologies and electrochemiluminescence detection to achieve ultra-fast, highly sensitive assays in a homogeneous format. Cellular membranes containing the EGF receptor were passively adsorbed to MSD proprietary coated electrodes embedded in multi-well plates. Binding of EGF to the EGF receptor was detected by inducing and measuring electrochemi- luminescence from a labeled EGF ligand. Approximately 1000 cell equivalents per well yielded a signal to background ratio of 20. The observed KD agrees with that reported in the literature and demonstrates that immobilization of the membranes and modification of the ligand do not alter the binding affinity. Binding specificity was confirmed with two inhibitors. The assay can be readily adapted to facilitate analysis of a broad array of receptor-ligand interactions. TM TM A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes TM Multi-Array TechnologyTM Multi-Array Technology Unified technology platform with instruments, plates and reagents for drug discovery for drug discovery. Combines the power of microarrays with the sensitivity of electrochemiluminescence Combines the power of microarrays with the sensitivity of electrochemiluminescence.96-, 384- and 1536 microplate formats 96-,Multi-Spot 384- TMand plates 1536 with microplate high density formats. -
Detection of Virus-Specific Immunoglobulins Using a Doubly Labeled Fluorescein- 125I Antibody A
JOURNAL OF CLINICAL MICROBIOLOGY, June 1976, p. 637-639 Vol. 3, No. 6 Copyright © 1976 American Society for Microbiology Printed in U.S.A. Detection of Virus-Specific Immunoglobulins Using a Doubly Labeled Fluorescein- 125I Antibody A. J. PARKINSON* AND J. KALMAKOFF Department ofMicrobiology, University of Otago, Dunedin, New Zealand Received for publication 17 February 1976 Commercially prepared fluorescein-labeled antihuman antibodies were la- beled with 125I and used to compare specific herpes simplex virus antibody titers as determined by indirect fluorescent antibody and radioimmunoassay tech- niques. Total virus-specific immunoglobulin and virus-specific immunoglobulin G titers did not vary by more than one twofold dilution when compared by the two methods. Efforts are being made to develop a reliable calf serum, penicillin (100 U/ml) streptomycin radioimmunoassay (RIA) for the detection of (100 ,ug/ml), and 0.1% bicarbonate, were in- virus-specific immunoglobulins, acceptable for fected with the isolated virus. Uninoculated use in diagnostic serology (1, 2, 5-8). The estab- monolayers were maintained as controls. When lishment of a satisfactory RIA depends on the infected monolayers showed 75% cytopathic use of antibody with both a high avidity and effect, both inoculated and uninoculated cells selectivity for the material to be assayed (4). were dispersed, using 0.015% ethylenediamine- Consequently, a major obstacle to using RIA tetraacetic acid. Both cellular suspensions were routinely is the necessity of preparing specific standardized to contain 2.5 x 105 cells/ml in high-titer antibody against human immuno- phosphate-buffered saline. Using 0.025-ml vol- globulins (IgG, IgM, and IgA).