Laboratory Methods
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Mcb 407-Immunology and Immunochemistry-Lecture Note
MCB 407-IMMUNOLOGY AND IMMUNOCHEMISTRY-LECTURE NOTE DR. D. A. OJO BRIEF HISTORICAL REVIEW OF IMMUNOLOGY The mechanism by which antibody are formed has been debated for years. It was proposed that the specificity of an antibody molecule was determined both by its amino acid sequence but by the molding of the peptide chain around the antigenic determinant. This theory lost favour when it became apparent that antibody-forming cells were devoid of antigen and that antibody specificity was a function of amino acid sequence. At present, the CLONAL (proposed by Burnete) SELECTION THEORY is widely accepted. It holds that an immunologically responsive cell can respond to only one antigen or a closely related group of antigens and that this property is inherent in the cell before the antigen is encountered. According to the clonal selection theory, each individual is endowed with a very large pool of lymphocytes, each of which is capable of responding to a different antigen. When the antigen enters the body, it selects the lymphocyte which has the best “fit” by virtue of a surface receptor. The antigen binds to this antibody-like receptor, and the cell is stimulated to proliferate and form a clone of cells. Thus, selected cells quickly differentiate into plasma cells and secrete antibody which is specific for the antigen which served as the original selecting agent (or a closely related group of antigens). The History of Blood Transfusion Man’s centuries-long desire to perform blood transfusion as a therapeutic procedure forms the cornerstone of the modern science of immunohematology. At present time, the use of whole blood is a well-accepted and commonly employed measure without which many modern surgical procedures could not be carried out. -
"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. -
TITAN IV Immunoelectrophoresis Procedure
TITAN IV Immunoelectrophoresis Procedure Cat. No. 9050 and 9061 Helena Laboratories Titan IV Immunoelectrophoresis (IEP) is intended for barbital-sodium barbital buffer with 0.1% sodium azide added semiquantitative determinations by immunoelectrophoresis. as a preservative. WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. This SUMMARY product contains sodium azide. Refer to the sodium azide Immunoelectrophoresis (IEP) combines two techniques, warning. electrophoresis and immunodiffusion. In this two-part procedure, Preparation for Use: The plates are ready for use as proteins in a serum or urine sample are first separated packaged. according to charge by electrophoresis. Then, antisera Storage and Stability: Plates should be stored flat, in the complimentary to the proteins under study are applied to the protective packaging, at 2°C to 8°C and are stable until the plate and allowed to diffuse. When a favorable antigen to expiration date indicated on the label. DO NOT FREEZE antibody ratio exists, a precipitin arc will form on the plate. PLATES OR EXPOSE THEM TO EXCESSIVE HEAT. IEP is used for the diagnosis and differential diagnosis of Signs of Deterioration: The plates should have a smooth, monoclonal gammopathies when using serum or urine clear agarose surface. Discard the plates if they appear 1-4 specimens. The method is also used for a number of other cloudy, show bacterial growth, or if they have been exposed to purposes, including screening for circulating immune freezing (a cracked or bubbled surface) or excessive heat (a complexes, characterization of cryoglobulinemia and dried, thin surface). pyroglobulinemia, recognition and characterization of antibody 2. Antisera for Assay of Immunoglobulins syndromes, and recognition and characterization of the various Antiserum to Human IgG, Cat. -
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). -
IMMUNOELECTROPHORESIS TEST Dr
[Type text] IMMUNOELECTROPHORESIS TEST Dr. Daljeet Chhabra Department of Veterinary Microbiology College of Veterinary Science and A.H., Mhow NDVSU, Jabalpur The term “immunoelectrophoresis” was first coined by Grabar and Williams in 1953. Immunoelectrophoresis refers to precipitation in agar under an electric field. It is a process of a combination of immuno-diffusion and electrophoresis. An antigen mixture is first separated into its component parts by electrophoresis and then tested by double immuno-diffusion. Antigens are placed into wells cut in a gel (without antibody) and electrophoresed. A trough is then cut in the gel into which antibodies are placed. The antibodies diffuse to meet diffusing antigens, and lattice formation and precipitation occur permitting determination of the nature of the antigens. Principle: When an electric current is applied to a slide layered with gel, the antigen mixture placed in wells is separated into individual antigen components according to their charge and size. Following electrophoresis, the separated antigens are reacted with specific antisera placed in troughs parallel to the electrophoretic migration and diffusion is allowed to occur. Antiserum present in the trough moves toward the antigen components resulting in the formation of separate precipitin lines in 18-24 hrs, each indicating reaction between individual proteins with its antibody. Materials required: Agarose gel, slides, well cutters, Immunoelectrophoresis machine, buffer, etc. Procedure: 1. Agarose gel is prepared on a glass slide put in a horizontal position. 2. After well cutting, sample is added in well. 3. The gel is placed into the electrophoresis chamber with the samples on the cathodic side, and electrophoresis runs for 20 mins/ 100 volts. -
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. -
The Power and Limitations of Influenza Virus Hemagglutinin Assays
ISSN 00062979, Biochemistry (Moscow), 2017, Vol. 82, No. 11, pp. 12341248. © Pleiades Publishing, Ltd., 2017. Original Russian Text © N. B. Ustinov, E. G. Zavyalova, I. G. Smirnova, A. M. Kopylov, 2017, published in Biokhimiya, 2017, Vol. 82, No. 11, pp. 15771592. REVIEW The Power and Limitations of Influenza Virus Hemagglutinin Assays N. B. Ustinov, E. G. Zavyalova*, I. G. Smirnova, and A. M. Kopylov Lomonosov Moscow State University, Faculty of Chemistry, 119991 Moscow, Russia; Email: [email protected] Received May 12, 2017 Revision received August 8, 2017 Abstract—Influenza virus hemagglutinins (HAs) are surface proteins that bind to sialic acid residues at the host cell surface and ensure further virus internalization. Development of methods for the inhibition of these processes drives progress in the design of new antiviral drugs. The state of the isolated HA (i.e. combining tertiary structure and extent of oligomerization) is defined by multiple factors, like the HA source and purification method, posttranslational modifications, pH, etc. The HA state affects HA functional activity and significantly impacts the results of numerous HA assays. In this review, we ana lyze the power and limitations of currently used HA assays regarding the state of HA. DOI: 10.1134/S0006297917110025 Keywords: influenza virus, surface antigens, influenza hemagglutinin, inhibitors of hemagglutination, monoclonal antibod ies, ELISA Influenza is one of the most common infectious dis endosome with subsequent nucleoprotein release from eases: 510% of adults and 2030% children are infected the endosome (HA and M2 protein), and detachment of with influenza each year. Influenza infection itself lasts the newly synthesized virus particles from the host cell only for several days, but the high risk for human health surface (neuraminidase, NA) [35]. -
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.