Immunochemical Techniques HSS 3109 PDF

HSS 3109 - Research Approaches in Health Biosciences Yan Burelle, Ph.D. Professor Interdisciplinary School of Health Sciences, Faculty of Health Sciences & Department of Cellular and Molecular Medicine, Faculty of Medicine University Research Chair in Integrative Mitochondrial Biology University of...

HSS 3109 - Research Approaches in Health Biosciences Yan Burelle, Ph.D. Professor Interdisciplinary School of Health Sciences, Faculty of Health Sciences & Department of Cellular and Molecular Medicine, Faculty of Medicine University Research Chair in Integrative Mitochondrial Biology University of Ottawa Pavillon Roger Guindon Room 2117 451 Smyth Road, Ottawa, Ontario K1N 8M5 Lab website : www.burellelab.com Phone (office) : 613-562-5800 ext 8130 Immunochemical techniques Learning objectives • Know how antibodies are used in research and clinics • Understand the basics of antibodies • Learn how antibodies are made • Understand the basis and the use of the following approaches in health biosciences: • Immuno assays • Immuno-microscopy • Epitope mapping • Immunoblotting • Immunoprecipitation • FACS Use of antibodies for research and clinical purposes Antibodies are all over the place in biomedical research biochemistry lab, patient etc. In the lab… Hospital Setting • Isolate or quantify the level of specific proteins • Locate the tissue and subcellular localization of specific proteins • Identify protein-protein interactions Immunoblot to look at contents of a specific protein that we are interested in Fluroercently labelled antibody - can see cytoskeleton network Immunopreciptiation use antibodies that recognize T4, insulin, glucagon, etc. Therapeutic • ex. Antibodies against the EGF receptor • Quantification of hormones • Vaccination etc. based on the • Diagnostics of infectious diseases COVID, presence of viral or bacterial • Diagnostics of genetic disorders proteins Level of some proteins might be profoundly affected The basics of antibodies • Antibodies are naturally produced by B-lymphocytes Cells of adaptive immune system The basics of antibodies • Mammals will produce antibodies to practically any foreign material that is introduced into their bodies providing it has a molecular weight greater than 5 000 Da. Those materials are antigens • Antibodies recognize specific regions on the antigen called epitopes which are usually composed of a sequence of 15 amino acids. • Different antibodies can be developed for the same antigen. Each antibody will recognize a specific epitope immiune system mounts response against these The basics of antibodies • In the body, antibodies are produced in lymphatic nodes • penetrates lymph node through lymphatic circulation • B cells recognizing specific antigens are activated by binding of the antigens to B-cell receptors • B-cells become activated and differenciate into plasmocytes which produce large amounts of antibodies. Full activation and differentiation of B cell into plasma cell B cell receptor without tail that normally anchors to the cell soluble B cell receptor factory to produce mass amount of antibodies The basics of antibodies • Antibodies are composed of two heavy chains and two light chains Tailored to specifically recognize antigen • The variable region contains the specific antigen binding site. The diversity of this region is quasi infinite which explains the high specificity of the antibodies • The constant region is divided in 5 different classes: IgM, IgG, IgA, IgE, IgD which have different biological activities And distinct immunological properties • IgG are the antibodies mainly used in immunochemical techniques • High specificity they bind better with lower concentration of antigens they’re supposed to detect • Present in large amounts in blood • Can be modified by labelling with marker molecules To make it visible, detectable, etc. without losing function Harnessing the immune system for antibody production • There are two major classes of antibodies used in immunochemistry: 1 ) polyclonal and 2) monoclonal. Polyclonal • Polyclonal antibodies are a population of antibodies binding different epitopes of the same antigen. that have initially reacted to the antigen • They are generated by different b cell clones each contributing one antigen-specific antibody • They are cheapest to produce, robust but less specific and more variable from batch to batch Since each time you produce a new batch of antibody, it will never be the same B lymphocyte producing the antibodies • • end up having various proportions of antibodies that recognize various epitopes, etc. there may be antibodies in this population that are poor at recognizing antigen - decrease batch quality • They are primarily used for research and diagnosis, not for therapy More chance of cross reactivity, etc. Harnessing the immune system for antibody production • Polyclonal antibodies are produced in animals by injecting them with antigens (usually 4-5 times) Inject antibody on first day with adjuvant to stimulate the IS to develop antibodies • every 20 days or so, inject a second or third dose - use incomplete peravdjuvant just to keep the immune system primed and maximize the output of the antibody production • at various time points, we will take blood draws and isolate the antibodies present in mass amounts in the blood of the animals • Antibodies are isolated from the animal blood serum and purified on affinity column • Larger animals are usually employed to obtained greater yield (rabbit, goat, donkey) Not mice or rats as they are too small Can produce antibodies later if they are re-exposed Ex. To produce human protein - design and inject amino acid sequence that is located in part of the Parkin gene that is easily accessible (ex. External shell) —> produce antiparkin antibodies (human form - will react and recognize human proteins) collected through blood draws Harnessing the immune system for antibody production Monoclonal • Monoclonal antibodies bind a single epitope on the antigen (vs a single antigen for polyclonal) • They are generated by a single b-cell clone called hybridoma • They are more expensive to produce but are more specific less batch to batch variation since it is the same cell producing the same antibody over and over again • Monoclonal antibodies are used in all fields including therapy. because of their specificity and reproducilibility between batches Harnessing the immune system for antibody production • Hybridomas are obtained by fusing single lymphocytes taken from the spleen of an immunized mice to a single cancer cell (myeloma cell) Start w/ mouse (not big animal) • Each hybridoma is tested for reactivity to the antigen • The clone producing the antibody with the best properties is selected and grown in large quantities. cancer cell line • divide rapidly and immortal, nice properties for culture Can they detect their antigen at low quantities (potency), is the antibody binding specifically to other stuff Take each one separately and test them • pick the one that works best Harnessing the immune system for antibody production Make them visibly enrich and purify specific antigens out of the mixture • Antibodies can be conjugated with a variety of molecules to allow: • Detection by colorimetric and fluorimetric methods • Immuno selection, immuno precipitation Immunoassays • The vast majority of immunoassays carried out fall into the category of enzyme immunosorbent assays • They are commonly used for: • the diagnosis of infectious agents such as viruses • The quantitative measurment of substances in blood such as hormones cytokines and chemokines • • used in research and hospital setting In this technique the antigenThe isbottom immobilised of 96 well plastic dish on to a solid phase, the most common being the enzyme linked immunosorbent assay (ELISA) plate. Immunoassays • Immobilisation of the antigen is achieved by the use of a coating or capture antibody • Detection is allowed by the addition of a second antibody (detector antibody) which is labelled with a reporter enzyme that generates a signal that is proportional to the amount of antigen bound to the solid phase. • Conventional ELISA can detect picomole amounts of antigens (10-12M) Typically in the range of circulating hormones that we have - insulin, glucagon, growth hormones, etc. antibody that is specific to the antigen to be detected and quantified • target antigen will stick at the bottom of the dish Basically same as capture antibody • detects Rinse - end up w/ and target antigen binds to and everything antigen else removed • good position to quantify when you add specific substrate for these enzymes, it will produce a colour • the darker the colour in the well, the more antigen was present in sample Most common form of ELISA - sandwich ELISA - antigen is in a sandwich between two antibodies Reporter antibody binds to detector antibody Immunoassays • There are different types of ELISA’s: • Direct ELISA • Indirect ELISA • Sandwich ELISA • Competitive ELISA Only difference to direct ELISA is secondary antibody that has detection system Inhibitor antigen - molecule you can add that can compute w/ antigen for the antibody • can prevent ELISA system from detecting antigen antigen is not immobilized with still no specificity in capture antibody - more chance capturing antigen that other antigens might also bind on pastic plate • only one antibody (detection antibody) The chances of non-specific binding or cross reactions its with other stuff is minimized • Sandwich ELISA is more specific vs the first two since the antigen has to bind to two antibodies to be detected Sandwich ELISA standard curve • Sandwich and competitive ELISAs are quantitative when used with a standard curve standard concentration and optical density at Put known amounts of antigen and do serial dilution 450 nanometers - wavelength to detect the signal perform reaction and look at amount of light being produced • good liar relationship • duplicates for more precision • from the equation, you can precisely • in other wells, we can put samples from patients and determine when you read the optical density. relate it to standard the concentration of antigen in unknown samples • more concentration more light more colour Immunoassays Competitive ELISA standard curve Antigen inhibitor only thing that varies is amount of target protein All other antibodies capture inhibitor w/ fluroesnt molecule Less space left for antigen inhibitor to bind Low amounts of target protein will produce a lot of signal/light signal will be weak Advantage: you can be more precise on the minute differences in the concentration across samples Immuno-microscopy • Immuno-microscopy uses antibodies coupled to a detection system to visualize specific molecules within tissues or cells • Direct labeling : 1 antibody (primary is also detector fluorsent molecule or HRP etc. • Indirect labeling: 2 antibodies (primary and detector) • Samples can be frozen or fixed (formaline, glutaraldehyde…) Set cell still and prevent degredation once they are dead • Cells need to be permeabilized for the antibodies to bind the intracellular epitopes if you don’t permeabilize cell - antibodies will not go inside cell • this is fine if antigen is a membrane protein • won’t work for intracellular or intranuclear proteins For proteins in cytoplasm For nucleus or inside organelles Immuno-microscopy Optical or fluorscent microscopy to detect antigen of interest Myosin heavy chain 7 in cardiac muscle revealed with HRP Mitochondrial TOM20 and PDH revealed by fluorescence confocal microscopy superimpose both images • see that mitochondria contains the two proteins Immuno-microscopy • Immunoelectron microscopy is a technique where antibodies conjugated to gold particles are used to visualise antigens at the nanometer scale Adapted to electron microscopy • electrons hit gold particles and deflect • black dots • Gold is electron-dense and is seen as a dark shadow against the light background of the specimen field. • Particles or different sizes can be used to track two different antigens on the same image (bottom image) TOM20 Much higher resolution PDH Epitope mapping important for designing antibodies that have a specific place you want them to bind to on the protein you want to look at - if you don’t know where epitope is, you don’t know where antibody will bind • Epitope mapping is the process of experimentally identifying the binding sites of antibodies on their target antigen. each piece - string of amino acid • This technique is an important component in the development of therapeutic monoclonal antibodies and vaccines used for the treatment of infectious diseases and cancer bind to epitope and generate a pattern • based on pattern you can identify the sequence of amino acid where the antibody binds and therefore the region of the epitope Ex. VGF receptor - promotes tumour growth - put antibody on outer part to block process • The overlaping peptide scan technique is a high troughput inexpensive approach to map epitopes. • An antibody is added to an array of synthetic overlapping peptides taken from an antigen • Binding is detected by reading the array signal Staggered - cover entire length of antigen Specific - one spot several signals Always entered around an amino acid +/- a few other ones around Immunoblotting • Antibodies are also used to quantify the amount of an antigen immobilized on a membrane support. • This technique known as immunoblotting or western blotting is covered in more details in the protein detection and analysis module Precipitate visible by eye Probably raised in goat or donkey Use electrophoresis to migrate protein on gel • migrates according to size of chemical properties • once seperated we can detect amount of a specific protein in a mixture • the darker the band, the more GAPPH Incubate membrane w/ primary antibody developed in mouse to detect human b-actin Immuno-precipitation immunocapture primary antibody with antigen bound to it Use antibody to immunocapture specific protein form mixture • couple antibody to magnetic bead or agarose bead to pin down and separate antibody form mixture Have secondary antibody conjugated at surface of bead Can spin down in centrifuge or if magnetic you can put a magnet at the bottom of the tube to pull it down and get rid of everything else from beads downstream analysis Release antigen • Antibodies are also used to purify an antigen present is a complex mixture • This technique known as immunoprecipitation is covered in more details in the protein detection and analysis module Fluorescence Assisted Cell Sorting (FACS) • Flow cytometryCountisindividual a laser-based technology cells employed in cell counting, cell sorting, biomarker detection and protein engineering. • Cells are suspended in a stream of fluid and passed through an electronic detection apparatus that counts single particles cell suspension • incubate cells with antibodies that recognize proteins at their surface (cellsurface proteins) • Size of cells can be detected by the analysis of forward light scatter (FCS) • Granularity or fluorescence level of an antibody labelled-cell is detected by measuring side scatter (SSC) Can count hundreds of thousands of cells in a short period side scatter “Postive or negatively fluroscently labelled” Pushes cells in tiny capillary - small enough to enter one by one infant of a laser beam - on other side there are two detectors Fluorescence Assisted Cell Sorting (FACS) • Flow cytometers allow simultaneous multiparametric analysis of the physical and molecular characteristics of up to thousands of particles per second. Charged deflector plates • When coupled to sorting devices FACS allow separation of various cell populations present in a mixture Each dot is a signal generated by a single cell • can see hundreds of thousands of cells here • draw a “gate” around these clouds of cells and ask the system to separate them into appropriate tubes • ex. Can use to isolate muscle stem cells CD4 positive, CD3 negative Direct to different tubes • ex. Sort cells that are all positive for a green fluorophore, red flurophor or cells that are positive for a green marker or a purple marker - enrich/seperate form a mixture Both CD4 and CD3 positive CD3 positive, CD4 negative Both CD3 and CD4 negative