A Transit Time-Resolved Microflow Cytometry-Based Agglutination
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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. -
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. -
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). -
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. -
Development and Evaluation of a Simple Latex Agglutination Test for Diagnosis of Tuberculosis' R
APPuED MICROBIOLOGY, Oct. 1972, p. 525-534 Vol. 24, No. 4 Copyright 0 1972 American Society for Microbiology Printed in U.S.A. Development and Evaluation of a Simple Latex Agglutination Test for Diagnosis of Tuberculosis' R. V. COLE,2 A. W. LAZARUS, AND H. G. HEDRICK Department of Botany and Bacteriology, College of Life Sciences, Louisiana Tech University, Ruston, Louisiana 71270 Received for publication 1 March 1972 A simple latex agglutination test (SLAT) based on modifications of existing serodiagnostic techniques, in which commercially available reagents are used, was developed for detection of antibodies against Mycobacterium tuberculosis. Tests performed on 553 serum samples from 316 individuals, including 117 bacteriologically confirmed active tuberculosis patients, showed 80% positive titers. Sera from 12 patients with arrested tuberculosis showed 91% positive titers. Nonspecific reactions were noted in 5% of 160 serums from selected normal individuals and patients with diseases other than tuberculosis. The an- tibodies detected by the SLAT method were found to be relatively stable when exposed to low temperatures, whereas high temperatures reduced the antibody titer considerably. Disodium ethylenediaminetetraacetic acid inactivation of serum complement was found to be satisfactory. No variation of tuberculosis antibody titer was noted in tests on multiple specimens from patients whose conditions were stabilized. However, considerable fluctuation was encountered in antibody titers obtained on recently detected individuals. Data obtained in this study indicate that the modified procedures of the SLAT method could replace the tuberculin skin test for simple screening of tuberculosis in adults. The use of tuberculin skin test procedures of serodiagnostic methods for the detection of has been the basis for the detection of new tuberculosis antibodies. -
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. -
Principles of Immunochemical Techniques Used in Clinical Laboratories
Review Received 2.11.06 | Revisions Received 3.1.06 | Accepted 3.2.06 Principles of Immunochemical Techniques Used in Clinical Laboratories Marja E. Koivunen, Richard L. Krogsrud (Antibodies Incorporated, Davis, CA) DOI: 10.1309/MV9RM1FDLWAUWQ3F Abstract binding site. The type of antibody and its diseases. Immunoassays can measure low Immunochemistry offers simple, rapid, robust affinity and avidity for the antigen determines levels of disease biomarkers and therapeutic or yet sensitive, and easily automated methods assay sensitivity and specificity. Depending on illicit drugs in patient’s blood, serum, plasma, for routine analyses in clinical laboratories. the assay format, immunoassays can be urine, or saliva. Immunostaining is an example Immunoassays are based on highly specific qualitative or quantitative. They can be used for of an immunochemical technique, which binding between an antigen and an antibody. the detection of antibodies or antigens specific combined with fluorescent labels allows direct An epitope (immunodeterminant region) on the for bacterial, viral, and parasitic diseases as visualization of target cells and cell structures. antigen surface is recognized by the antibody’s well as for the diagnosis of autoimmune Immunochemistry offers simple, rapid, robust yet sensitive, bind to an antigen. The third domain (complement-binding Fc and in most cases, easily automated methods applicable to routine fragment) forms the base of the Y, and is important in immune analyses in clinical laboratories. Immunochemical methods do not system function and regulation. usually require extensive and destructive sample preparation or The region of an antigen that interacts with an antibody is expensive instrumentation. In fact, most methods are based on called an epitope or an immunodeterminant region. -
Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay Stephanie D
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector RESEARCH TECHNIQUES MADE SIMPLE Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay Stephanie D. Gan1 and Kruti R. Patel2 Journal of Investigative Dermatology (2013) 133, e12. doi:10.1038/jid.2013.287 INTRODUCTION Enzyme immunoassay (EIA) and enzyme-linked immunosor- WHAT ENZYME-LINKED bent assay (ELISA) are both widely used as diagnostic tools in IMMUNOSORBENT ASSAY (ELISA) DOES medicine and as quality control measures in various industries; • ELISA is a biochemical assay that uses antibodies they are also used as analytical tools in biomedical research and an enzyme-mediated color change to detect for the detection and quantification of specific antigens or anti- the presence of either antigen (proteins, peptides, bodies in a given sample. These two procedures share similar hormones, etc.) or antibody in a given sample. basic principles and are derived from the radioimmunoassay (RIA). RIA was first described by Berson and Yalow (Yalow and • Both “indirect” and “sandwich” ELISAs allow Berson, 1960), for which Yalow was awarded the Nobel Prize detection of antigen or antibody at very low in 1977, to measure endogenous plasma insulin. RIA was then concentrations. developed into a novel technique to detect and measure bio- • The competitive method detects compositional logical molecules present in very small quantities, paving the differences in complex antigen mixtures with way for the analysis and detection of countless other biologi- high sensitivity, even when the specific detecting cal molecules, including hormones, peptides, and proteins. antibody is present in relatively small amounts. -
Rheumatoid Arthritis
Ann Rheum Dis: first published as 10.1136/ard.38.3.248 on 1 June 1979. Downloaded from Annals of Rheumatic Diseases, 1979, 38, 248-251 The antiperinuclear factor. 1. The diagnostic significance of the antiperinuclear factor for rheumatoid arthritis INEZ R. J. M. SONDAG-TSCHROOTS, C. AAIJ, J. W. SMIT, AND T. E. W. FELTKAMP From the Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, and the Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, The Netherlands SUMMARY In 1964 Nienhuis and Mandema reported the presence of antibodies against cytoplasmic granules in buccal mucosal cells in the serum of 50% of patients with rheumatoid arthritis (RA). Although they reported a good specificity for RA of these so-called antiperinuclear antibodies (APF), their results never threatened the monopoly of the rheumatoid factor as a serological tool for the diagnosis of RA. A re-evaluation with improved immunofluorescence methods showed a frequency of the APF of 78 % in 103 patients with RA. The latex test and the Waaler-Rose test were positive in only 7000 and 580% respectively of these patients. Only 150% of the RA patients were negative for all 3 tests. Thus, 400% of patients who were seronegative by the traditional methods gave a positive result on performance of the APF test. The high sensitivity of the APF test was combined with a good specificity, for the frequency in patients with other autoimmune diseases or degenerative joint disease and in healthy subjects was low. For the serodiagnosis of RA it seems best to combine the use of the APF test with one for rheumatoid factor. -
1 Supplemental Methods Isolation of Total Protein, Soluble Nuclear And
Supplemental methods Isolation of total protein, soluble nuclear and chromatin fraction After washing the cells twice with PBS, total protein extracts were isolated in RIPA buffer (1× PBS, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxychelate, 0.1% (w/v) SDS) supplemented with 1 mM Pefabloc and 1 ng/µl Aprotinin/Leupeptin. Preparation of soluble and insoluble nuclear extract was performed as described (10). Briefly, cells were washed twice with PBS before resuspending in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% (v/v) glycerol, 1 mM DTT, 0.1% (v/v) Triton X-100, supplemented with protease inhibitors: 1 mM Pefabloc, 1 ng/µl Aprotinin/Leupeptin and 10 mM β-glycerophosphate) and incubated on ice for 5 minutes. By centrifuging at 1,300×g for 5 minutes at 4 °C nuclei were isolated, washed once with buffer A (depleted of Triton X- 100) and subsequently lysed in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT plus supplements as in buffer A). Soluble and insoluble (chromatin) fraction were separated via centrifugation at 1,700×g for 4 minutes at 4 °C. Chromatin samples were subsequently resuspended in buffer B. Apoptosis assay Apoptosis was analyzed using the Guava Nexin® assay (Guava Technologies, Millipore) according to the manufacturer’s instructions. Briefly, trypsinized cells were collected, centrifuged and the cell pellets were resuspended in 500 µl of medium. After diluting the cells to a concentration of 2× 105 - 1× 106 cells/ml, 100 µl of each diluted cell suspension and 100 µl of Guava Nexin solution were mixed.