DOCSLIB.ORG

A Novelquantitative Immunoassay System for P53 Using Antibodies

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Novelquantitative Immunoassay System for P53 Using Antibodies J Clin Pathol 1997;50:143-147 143 A novel quantitative immunoassay system for p53 using antibodies selected for optimum designation J Clin Pathol: first published as 10.1136/jcp.50.2.143 on 1 February 1997. Downloaded from of p53 status M D Thomas, G G McIntosh, J J Anderson, D M McKenna, A H Parr, R Johnstone, T W J Lennard, C H W Horne, B Angus Abstract The prediction of likely outcome in cancer Aim-To develop a highly sensitive and would permit allocation of patients to appro- specific enzyme linked immunosorbent priate aggressive therapeutic regimens in in- assay (ELISA) system for analysis of p53 stances where prognosis is poor; and con- protein in cancer lysates. versely, patients with good prognosis could be Methods-The anti-p53 monoclonal anti- spared dangerous and debilitating treatment. bodies D07, 1801, BP53.12, and 421, and Mutations of the tumour suppressor gene p53 anti-p53 polyclonal antiserum CM1 were can result in dysfunction, with accumulation of assessed by immunohistochemistry and the inactive/mutated protein which increases genomic instability leading to the expression western blot analysis to identify those and progression of the metastatic phenotype.' most suitable for determining p53 status Accumulation ofp53 has been shown in several of cancer celis. Antibodies with desired different cancers.2" In breast cancer, we and characteristics were used to develop a others have shown that accumulation of p53 is non-competitive sandwich type ELISA associated with poor clinical prognosis.57 system for analysis of p53 expression in Although p53 overexpression can be detected cancer cytosols. Using the ELISA, p53 immunohistochemically, only a qualitative esti- protein concentrations were measured in mation can be made. It has, however, been a small series of breast cancers, and the proposed that quantitative analysis of p53 pro- quantitative values compared with p53 tein concentrations in cancer will provide addi- immunohistochemical data of the same tional objective prognostic information.8 Sev- cancers. eral groups have described non-competitive Results-DO7 and 1801 gave the most sandwich type immunological based assay sys- specific and reliable results on immuno- tems, using monoclonal/polyclonal antibody http://jcp.bmj.com/ histochemisry and western blot analysis. combinations, for quantitative analysis of p53 Using these two antibodies, a non- in tumour cytosol.9-' Using immunohisto- competitive sandwich type ELISA system chemistry, we have recently reported that was developed to p53 assessment of p53 status in breast cancer tissue analyse quantita- sections is critically dependent upon the tively. Analysis of the breast cancer series antibodies used for screening, and influences showed a good correlation between immu- the significance of the prognostic data on September 23, 2021 by guest. Protected copyright. nohistochemistry and the ELISA- generated.5 Similarly, the choice of antibodies tumours were generally positive using to be applied in quantitative p53 assays may both techniques. Discrepancies were exert a notable effect on the final estimate of noted however: some cancers were immu- tumour p53 concentrations. Selection of the Department of nohistochemically negative but ELISA Pathology, Royal most appropriate antibodies will assure the Victoria Infirmary, positive. One explanation for this may be specificity and reproducability of the data on University of that the ELISA is more sensitive than which subsequent assessments of patient prog- Newcastle upon Tyne, immunohistochemistry. nosis may be based. Newcastle upon Tyne Conclusion-The p53 ELISA system is a NEl 4LP double monoclonal M D Thomas non-competitive anti- Methods sandwich G G McIntosh body method, using D07 and TUMOUR TISSUE J J Anderson 1801 which have been shown to be highly Samples of mammary carcinoma tissue were D M McKenna specific for p53 protein by immunohisto- collected at surgery. The tumour was dissected A H Parr and western blot The R Johnstone chemistry analysis. free of superfluous tissue and representative C H W Horne lower threshold of the assay is 0.1 ng/nd samples processed for the production of frozen B Angus analyte in an enriched recombinant p53 sections and cytosols. preparation. As p53 is now regarded as a Department of Clinical protein associated with prognosis in Surgery CYTOSOL PREPARATION T W J Lennard breast and other cancers, the assay may p53 was quantified in cytosol extracts prepared have clinical applications. for routine estimation of oestrogen/ Correspondence to: (7 Clin Pathol 1997;50:143-147) progesterone receptor analysis. Frozen tumour Dr B Angus. tissue (approximately 5 mm2) was powdered Accepted for publication Keywords: p53; p53 antibodies; immunohistochemis- using a micro-dismembranator and resus- 5 November 1996 try; western blot; quantitative ELISA. pended in 1 ml HEPES/EDTA buffer (20 mM 144 Thomas, McIntosh, Anderson, McKenna, Parr, J7ohnstone, et al HEPES, 1.5 mM EDTA, pH 7.4) containing WESTERN BLOT ANALYSIS protease inhibitors: aprotinin (2 jg/ml), leu- Specificity of the p53 antibodies was assessed J Clin Pathol: first published as 10.1136/jcp.50.2.143 on 1 February 1997. Downloaded from peptin (2 jg/ml), phenylmethylsulphonyl fluo- using western blot analysis against a cell lysate ride (PMSF) (50 jig/ml), and pepstatin (1 jg/ of the breast adenocarcinoma cell line MDA- ml). Further disruption was achieved using MB-231. Cells mechanically harvested from sonication. Owing to the heat lability of the confluent 250 ml tissue culture flasks were proteins to be assayed, the samples were main- washed three times with PBS and lysed in tained on ice throughout the process. The 1.5 ml ice cold Laemmli buffer (0.125 M Tris, tissue/cell lysate was clarified by ultracentrifu- 4% SDS, 40% glycerol,1 % bromophenol blue, gation, 50 000 x g at 4°C for 40 minutes. and 5% mercaptoethanol, pH6.8). The lysate Cytosol fractions were collected, and stored at was held on ice, sonnicated using a single five -70°C prior to analysis for p53. Samples were second burst, and stored at -70°C until analysed within two weeks of preparation. required. Samples of lysate were fractionated by discontinuous SDS-polyacrylamide gel electrophoresis, using a 10% T, 2.6% C, poly- ESTIMATION OF TOTAL PROTEIN CONCENTRATION acrylamide gel. Proteins were transferred onto Total protein concentration of the cytosol nitrocellulose membrane using a semi-dry preparation was determined in relation to electroblotting system. Blots were probed with bovine serum albumin (BSA) using bicin- p53 antibodies D07, 1801, BP53.12, 421, choninic acid (BCA) protein assay kits (Pierce each at 0.5 jig/ml and with CM1 at dilutions of and Warriner Ltd). 1 in 1000 and 1 in 2000. Reacted p53 antibod- ies were detected using alkaline phosphatase conjugated goat anti-mouse immunoglobulin p53 ANTIBODIES or goat anti-rabbit immunoglobulin polyclonal Anti-p53 monoclonal antibodies D07, 1801 anti-sera. Blots were developed with 0.1 mg/ml and BP53.12, and the rabbit anti-p53 polyclo- nitroblue tetrazolium/0.15 mg/ml BCIP (5- nal antiserum CM1 were all obtained from bromo-4-chloro-3-indolyl phosphate), in de- Novocastra Laboratories Ltd, Newcastle upon velopment buffer (0.1 M Tris buffer, 0.1 M Tyne, UK. Anti-p53 monoclonal antibody 421 NaCl and 50 mM MgCl2, pH 9). was obtained from Oncogene Science Ltd (Sera Laboratories Ltd, Crawley Down, UK). SELECTION OF p53 ANTIBODIES FOR ELISA The concentration of monoclonal antibodies DEVELOPMENT 1801, D07, and BP53-12 was determined p53 antibodies for the development of the using IgG radial immunodiffusion. ELISA were selected on the basis of their per- formance in immunohistochemistry, for opti- IMMUNOHISTOCHEMISTRY mum assignment of p53 status, and their To assess the immunohistochemical perform- specificity for p53 in western blot analysis. http://jcp.bmj.com/ ance of antibodies directed against p53 for the Using these criteria, monoclonal antibodies assignment of p53 status, we stained serial 1801 and D07 were selected for use in assay cryostat sections of breast cancers with test development. monoclonal antibodies D07, 1801, BP53.1 and 421, and the rabbit polyclonal antiserum PREPARATION OF BIOTINYLATED Do7 CM1. Sections, 3 jim thick, of cryopreserved Monoclonal antibody D07 was purified from hybridoma culture supernatant by absorption on September 23, 2021 by guest. Protected copyright. tumour tissue were cut and air dried onto with protein A (Sigma). Antibody was eluted at microscope slides. Endogenous tissue peroxi- pH 3 and dialysed against 0.1 M borate buffer, dase activity was quenched using 0.6% hydro- pH 8.8. Purified antibody was then reacted gen peroxide in methanol, for five minutes. with ester of biotinamidocaproate N-hydroxy- Subsequently, non-specific immunostaining succinimide, in dimethyl sulphoxide (DMSO), was blocked by incubating the sections with (100 jg ester/mg monoclonal antibody) for 5% lamb serum/phosphate buffered saline four hours at room temperature. The conjuga- (PBS) for 30 minutes. Test p53 monoclonal tion reaction was terminated by the addition of antibodies at 0.5 jg/ml, or rabbit anti-serum 1 M ammonium chloride (259 l/mg mono- CM1 at a dilution of 1 in 1000 were applied clonal antibody), and the products extensively and incubated with sections for one hour at dialysed against 0.1 M Tris/HCl, pH 7.4. room temperature. Binding of p53 antibody was detected using biotinylated goat anti- ELISA PROCEDURE mouse or goat anti-rabbit polyclonal anti- A sandwich type ELISA system was developed serum and streptavidin/biotinylayed horseradish for the quantitative analysis of p53. Solid phase peroxidase complex (Dako, High Wycombe, monoclonal antibody 1801 was used as the p53 UK). Sections were developed with 3,3- immobiliser, and biotinylated D07 as the p53 diaminobenzidine tetrahydrochloride (Sigma, detector. In brief, Immulon-1 96-well ELISA Poole, Dorset, UK) in Tris buffered saline trays (Dynatech Laboratoies, Billinghurst, UK) (TBS) containing 0.3% hydrogen peroxide, were coated overnight at 4°C with 50 jl/well counterstained with Meyer's haematoxylin, de- 10 jg/ml 1801 in PBS.
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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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).
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • Radial Immunodiffusion Assay Protocol
    Radial Immunodiffusion Aim: To study the immunodiffusion technique by Single Radial Immunodiffusion. Introduction: Single Radial Immunodiffusion, also known as Mancini technique, is a quantitative immunodiffusion technique used to detect the concentration of antigen by measuring the diameter of the precipitin ring formed by the interaction of the antigen and the antibody at optimal concentration. In this method the antibody is incorporated into the agarose gel whereas the antigen diffuses into it in a radial pattern. Thus, the antibody is uniformly distributed throughout the gel. Principle: Single Radial Immunodiffusion is used extensively for the quantitative estimation of antigen. Here the antigen-antibody reaction is made more sensitive by the addition of antiserum into the agarose gel and loading the antigen sample in the well. As the antigen diffuses into the agarose radially in all directions, it’s concentration continuously falls until the equivalence point is reached at which the antigen concentration is in equal proportion to that of the antibody present in the agarose gel. At this point ring of precipitation (‘precipitin ring’) is formed around the well. The diameter of the precipitin ring is proportional to the concentration of antigen. With increasing concentration of antigen, precipitin rings with larger diameter are formed. The size of the precipitin rings depends on: Antigen concentration in the sample well Antibody concentration in the agarose gel Size of the sample well Volume of the sample Thus, by having various concentrations of a standard antigen, standard curve can be obtained from which one can determine the amount of an antigen in an unknown sample. Thus, this is a quantitative test.
    [Show full text]
  • 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.
    [Show full text]
  • Importance of Ag-Ab Reactions
    Ag-Ab reactions Tests for Ag-Ab reactions EISA SALEHI PhD. Immunology Dept. TUMS Importance of Ag-Ab Reactions • Understand the mechanisms of defense • Abs as tools in: – Treatment – Diagnosis • As biomarkers • As tools to measure analytes Nature of Ag/Ab Reactions http://www.med.sc.edu:85/chime2/lyso-abfr.htm • Lock and Key Concept • Non-covalent Bonds – Hydrogen bonds – Electrostatic bonds – Van der Waal forces – Hydrophobic bonds • Multiple Bonds • Reversible Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000 Affinity • Strength of the reaction between a single antigenic determinant and a single Ab combining site High Affinity Low Affinity Ab Ab Ag Ag Affinity = ( attractive and repulsive forces Calculation of Affinity Ag + Ab ↔ Ag-Ab Applying the Law of Mass Action: [[gAg-Ab] Keq = [Ag] x [Ab] Avidity • The overall strength of binding between an Ag with many determinants and multivalent Abs 4 6 10 Keq = 10 10 10 Affinity Avidity Avidity SifiitSpecificity • The ability of an individual antibody combining site to react with only one antigenic determinant. • The ability of a population of antibody molecules to react with only one antigen. Cross Reactivity • The ability of an individual Ab combining site to react with more than one antigenic determinant. • The ability of a population of Ab molecules to react with more than one Ag Cross reactions Anti-A Anti-A Anti-A Ab Ab Ab Ag A Ag B Ag C Shared epitope Similar epitope Factors Affecting Measurement of A/AbRAg/Ab Reac tions • Affinity • Avidity Ab excess Ag excess • AAbiAg:Ab ratio •Phyygsical form of Ag Equivalence – Lattice formation Do you need to know what happens in Lab.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]