Immunoassay - Elisa
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"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. -
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). -
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 . -
Development and Technical Validation of an Immunoassay for the Detection of APP669–711 (Aβ−3–40) in Biological Samples
International Journal of Molecular Sciences Article Development and Technical Validation of an Immunoassay for the Detection of APP669–711 (Aβ−3–40) in Biological Samples Hans W. Klafki 1,* , Petra Rieper 1, Anja Matzen 2, Silvia Zampar 1 , Oliver Wirths 1 , Jonathan Vogelgsang 1 , Dirk Osterloh 3, Lara Rohdenburg 1, Timo J. Oberstein 4 , Olaf Jahn 5 , Isaak Beyer 6, Ingolf Lachmann 3, Hans-Joachim Knölker 6 and Jens Wiltfang 1,7,8 1 Department of Psychiatry and Psychotherapy, University Medical Center (UMG), Georg-August-University, D37075 Göttingen, Germany; [email protected] (P.R.); [email protected] (S.Z.); [email protected] (O.W.); [email protected] (J.V.); [email protected] (L.R.); [email protected] (J.W.) 2 IBL International GmbH, Tecan Group Company, D-22335 Hamburg, Germany; [email protected] 3 Roboscreen GmbH, D-04129 Leipzig, Germany; [email protected] (D.O.); [email protected] (I.L.) 4 Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, D-91054 Erlangen, Germany; [email protected] 5 Max-Planck-Institute of Experimental Medicine, Proteomics Group, D-37075 Göttingen, Germany; [email protected] 6 Faculty of Chemistry, Technische Universität Dresden, D-01069 Dresden, Germany; [email protected] (I.B.); [email protected] (H.-J.K.) 7 German Center for Neurodegenerative Diseases (DZNE), D-37075 Göttingen, Germany 8 Neurosciences and Signaling Group, Institute of Biomedicine (iBiMED), Department of Medical Sciences, University of Aveiro, 3810-193 Aveiro, Portugal * Correspondence: [email protected] Received: 7 August 2020; Accepted: 2 September 2020; Published: 8 September 2020 Abstract: The ratio of amyloid precursor protein (APP)669–711 (Aβ 3–40)/Aβ1–42 in blood plasma was − reported to represent a novel Alzheimer’s disease biomarker. -
Allergy Skin and Challenge Testing
Corporate Medical Policy Allergy Skin and Challenge Testing File Name: allergy_skin_and_challenge_testing Origination: 7/1979 Last CAP Review: 11/2020 Next CAP Review: 11/2021 Last Review: 11/2020 Description of Procedure or Service Management of the allergic patient may include identifying the offending agent by means of allergy testing. Allergy testing can be broadly grouped into in vivo and in vitro methodologies: • In vivo testing - includes allergy skin testing such as the scratch, puncture or prick test (epicutaneous), intradermal test (intracutaneous) and patch test, and food and bronchial challenges. • In vitro testing - includes various techniques to test the blood for the presence of specific IgE antibodies to a particular antigen. Once the agent is identified, treatment is provided by avoidance, medication or immunotherapy (allergy shots). Allergic or hypersensitivity disorders may be manifested by generalized systemic reactions as well as by localized reactions in any organ system of the body. The reactions may be acute, subacute, or chronic, immediate or delayed, and may be caused by numerous offending agents: pollen, molds, dust mites, animal dander, stinging insect venoms, foods, drugs, etc. This policy only addresses in vivo (skin and challenge) testing; for vitro testing, see policy titled, Allergen Testing. Related Policies: Allergy Immunotherapy Desensitization Allergen Testing AHS – G2031 Maximum Units of Service Idi ***Note: This Medical Policy is complex and technical. For questions concerning the technical language and/or specific clinical indications for its use, please consult your physician. Policy BCBSNC will provide coverage for Allergy skin and challenge testing when it is determined to be medically necessary because the medical criteria and guidelines shown below are met. -
Guidance and Protocol for the Serological Diagnosis of Human Infection with Bordetella Pertussis
TECHNICAL DOCUMENT Guidance and protocol for the serological diagnosis of human infection with Bordetella pertussis As part of the EUpert-Labnet surveillance network www.ecdc.europa.eu ECDC TECHNICAL DOCUMENT Guidance and protocol for the serological diagnosis of human infection with Bordetella pertussis As part of the EUpert-Labnet surveillance network This report was commissioned by the European Centre for Disease Prevention and Control (ECDC), coordinated by Adoracion Navarro Torne, and produced by the members of the European Bordetella expert group ‘EUpert-Labnet’ as part of the project contract: Coordination of activities for laboratory surveillance of whooping cough in Member States and EEA countries (OJ/26/05/2011-PROC/2011/037) Authors Marion Riffelmann, HELIOS Kinikum Krefeld, Germany; Carl Heinz Wirsing von König, HELIOS Kinikum Krefeld, Germany; Dorothy Xing, National Institute for Biological Standards and Control (NIBSC), Health Protection Agency, Potters Bar, United Kingdom; Qiushui He, National Institute for Health and Welfare (THL), Turku, Finland. This protocol is intended to serve as a starting point for laboratories aiming at introducing ELISA serology for diagnosis of human Bordetella pertussis infections. Comments, requests or questions can be addressed to the authors. Version 1.0, October 2012 Disclaimer: this technical guidance is based on the latest available published data on diagnostic test performance at the time of writing. ECDC does not endorse any particular commercial product or instrument. Suggested citation: -
UBI® SARS-Cov-2 ELISA INSTRUCTIONS for USE
UBI® SARS-CoV-2 ELISA INSTRUCTIONS FOR USE FOR IN VITRO DIAGNOSTIC USE ONLY FOR EMERGENCY USE AUTHORIZATION ONLY FOR PRESCRIPTION USE ONLY INTENDED USE The UBI® SARS-CoV-2 ELISA is an Enzyme-Linked Immunosorbent Assay (ELISA) intended for qualitative detection of IgG antibodies to SARS-CoV-2 in human serum and plasma (sodium heparin or dipotassium (K2) EDTA). The UBI® SARS-CoV-2 ELISA is intended for use as an aid in identifying individuals with an adaptive immune response to SARS-CoV-2, indicating recent or prior infection. At this time, it is unknown for how long antibodies persist following infection and if the presence of antibodies confers protective immunity. The UBI® SARS-CoV-2 ELISA should not be used to diagnose or exclude acute SARS-CoV-2 infection. Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C 263a, that meet requirements to perform high complexity testing. Results are for the detection of IgG SARS CoV-2 antibodies. IgG antibodies to SARS-CoV-2 are generally detectable in blood several days after initial infection, although the duration of time antibodies are present post-infection is not well characterized. Individuals may have detectable virus present for several weeks following seroconversion. Laboratories within the United States and its territories are required to report all results to the appropriate public health authorities. The sensitivity of the UBI® SARS-CoV-2 ELISA early after infection is unknown. Negative results do not preclude acute SARS-CoV-2 infection. If acute infection is suspected, direct testing for SARS-CoV-2 is necessary. -
FOR REFERENCE USE ONLY: DO NOT USE in Place of Package Inserts Provided with Each Product
[US] • Isotonic saline solution Coombscell-E IgG-coated Red Blood Cells for the control of the antiglobulin Coombscell-E is a single vial of group 0 red blood cells sensitized with human test monoclonal IgG antibodies (specificity anti-D). • Reagent Red Blood Cells: Biotest: Biotestcell® 1 & 2 [REF] 816014100, FOR IN-VITRO DIAGNOSTIC USE ® Biotestcell® 3 [REF] 816085100, Biotestcell -I 8 [REF] 816020100, For tube test ® Biotestcell -I 11 [REF] 816021100 Package size • Donor or patient red blood cells [REF] 816030100 [VOL] 10 mL Coombscell-E • MLB 2 (Modified LISS Biotest) [REF] 8065200100 • Anti-Human Globulin Anti-IgG [REF] 804175100 • Anti-Human Globulin Anti-IgG, -C3d Polyspecific [REF] 804115100 Intended Use Coombscell-E Red Blood Cells are used for • Glass tubes 10 x 75mm or 12 x 75mm • in-house quality control (reactivity control of Anti-Human-Globulin) • Serological centrifuge • to control the technique of antiglobulin-test with negative results • Interval Timer to verify the negative results of the IAT (Indirect Antiglobulin Test) and DAT • Markers (Direct Antiglobulin Test) • Optical aid (optional). The use of an optical aid for agglutination reading must be vaildated by the user. Summary Moreschi first described the use of Anti-Human Globulin in 19081. Coombs Test procedure rediscovered the test in 1945.2,3 By injecting rabbits with human IgG, they A. Tube test were able to produce a protein (Anti-IgG) that reacted with incomplete antibodies (IgG). Most “incomplete” antibodies (IgG) fail to agglutinate red Verification of negative result in antiglobulin test blood cells suspended in saline.4 Most clinically significant antibodies in red 1.