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Immunochemical methods Generation and use of antibodies and antibody-based reagents for: •Detection •Quantification •Localization •Purification Henrik Wernérus [email protected] Antibodies (immunoglobulins) • Proteins capable of binding a molecule in a highly specific manner • Generated by the immunesystem in response to foreign substance • Important in vivo for inhibition or "labelling for destruction" of foreign substances ALSO: • Can be generated to almost any molecule for diagnostic, therapeutic or research interest 1 The antibody molecule Fab Binding function Fv (scFv) Fc H C 1 V L V C DR 3 CD R C 2 D R 1 Effector function CH 2 CH 2 CH 3 CH 3 L C C L 1 CH H Fv Fab IgG Fc Variablity located in the CDR regions • The sequence variability of the VL and VH domains is concentrated in three hypervariable regions that form the antigen-binding site. •Also called complementarity-determining regions (CDRs) •The remainder of the VL and VH domains form the framework regions (FRs) 2 Structure of the immunoglobulin fold • Diagram of an immunoglobulin light chain showing the structure of its constant (CL) and variable (CV) domains. •The CDR loop regions are shown in blue •The β-pleated sheets are held together by hydrophobic interactions and conserved disulfide bonds. The nature of antibody-antigen interactions •Cooperative effect from numerous attractive forces •Non-covalent interactions operates over a very small distance (1Å) •Shape complementarity is important! Basis for the specificity of antibody-antigen interactions. 3 Interaction between antibody and antigen • Interaction between antibody and influenza virus antigen (Coleman and Tulip, 1993) Generation of antibody subfragments by enzymatic digestion •For some applications the use of antibody fragments rather than the intact molecule are favoured. •Prevent binding to Fc receptors on the cell Surface (leukocytes) •Pepsin, Papain are the most commonly used proteolytic enzymes for generation of antibody fragments 4 Generation of polyclonal antibodies Antigen Immunization Blood Blood cells Plasma Coagulation factors Antiserum Enrich antibodies (specific or unspecific) Antigen Protein A/G Class specific methods (ex. Anti-IgE antibodies) General characteristics of the antibody response General characteristics of all specific immune responses A. Self/non-self discrimination B. Memory C. Specificity 5 Immunogen + Adjuvant Immune response Immunogen Immune response Adjuvants An adjuvant enhances the immune response upon immunization through one or several mechanisms including: • providing a slow-release of immunogen (depot effect) • stimulating T-cell help • beneficial presentation of immunogen Examples of commonly used adjuvants Adjuvant Composition and use Freunds complete adjuvant (FCA) Mineral oil containing heat-killed mycobacteria. Used as emulsion with aqueous antigen Used as emulsion with aqueous antigen Freunds incomplete adjuvant (FIA) Mineral oil Used as emulsion with aqueous antigen Alum Complex aluminium salts. There are various versions of the adjuvant: some can be purchased ready for use (e.g. Alhydrogel); others can be prepared in the laboratory by mixing various salts. Aqueous antigen is absorbed to gel. Quil A Saponin derived from Quillaja saponana Molina (South american tree). Mixed to form a complex with aqueous antigen (ISCOMS)....... Bacillus pertussis Killed organism mixed with aqueous antigen FCA/FIA is probably the most potent adjuvant but may be inappropriate for some purposes. 6 Antibodies from chickens (IgY) Chickens (birds) are evolutionary more distant from humans than mice or rabbits, making it more easy to obtain an immune response towards well conserved antigens. A single chicken can produce high amounts of antibody, up to 3 grams of IgY per month, which is 10-20 times the amount of a rabbit Antibodies are present in the yolk of the egg. Compared to rabbits, chickens produce antibody much quicker—high-titre antibody is available from eggs as early as day 25. Monoclonal vs Polyclonal antibodies •The polyclonal antiserum produced in response to a complex antigen contains a mixture of antibodies each specific for one of the epitopes present on the antigen. •A monoclonal antibody, derived from a single plasma cell, is specific for one epitope on a complex antigen. 7 Purification of antibodies •Some immunochemical techniques are frequently carried out using unpurified antibodies. •Often partial or complete purification of specific antibodies is required for use in immunochemical assays. Ligands for affinity chromatography: Ligand type: Examples(s): Antibody purification: Hapten DNP Antibodies that bind hapten Antigen Haemoglobin, FVIII Polyclonal antibodies of a single specificity Bacterial immunoglobulin binding proteins Protein A, Protein G Most IgG subclasses from many species Anti-immunoglobulin antibodies Goat anti-human IgG Class and/or species-specific IgG fraction Lectins Jacalin Mannan-binding Human IgA Mouse IgM Precipitation reactions (Antibody-antigen reactions) •The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments •The antigen must have at least two copies of the same epitope or have different epitopes that reacts with different antibodies present in polyclonal antisera 8 Immunodiffusion methods Radial immunodiffusion (Mancini method) The square of the precipitin ring diameter is proportional to the antigen concentration Double immunodiffusion(Ouchterlony method) Qualitative tool for determining the relationship between antigens and antibody Shared epitopes? Agglutination Antibody mediated precipitation of cells or particles Agglutination can be used for blood typing by mixing red blood cells with blood type specific antibodies 9 Species-specific antibodies Species-specific antibodies Important reagents in immunotechnology applications Isolate mouse antibodies Inject into goat (total pool, specific isotypes or fragments) Recover serum Other examples: • Rabbit anti-mouse IgG • Rat anti-mouse IgG1 • Sheep anti-rat IgM Purify goat-anti mouse antibodies Antibody labelling E Isotope Fluorophore Chromogenic substrate Product •Soluble •Precipitate F Horseradish Peroxidase (HRP) Alkaline Phosphatase (AP) β-Galactosidase (β-gal) Fluorescein (FITC) Rhodamine Phycoerythrin Cy-3 Cy-5 Alexa Fluor 125Iodine Gold Ferritin Wolfram ELISA Immunohistochemistry Immunoblotting Immunohistochemistry Immunocytochemistry Flow cytometry Immunoblotting Protein arrays Radioimmunoassays (RIA) Immunoelectron microscopy 10 Direct and indirect immunochemical assays The direct labeling method: • one step method • a specific antibody is complexed directly to the visible marker. Indirect labeling method: • two-step method • a primary antibody specific for the antigen • a secondary antibody coupled to a visible marker (enzymatic, fluorescent, gold) Immunoradioassay (RIA) • Competitive binding radioimmunoassay • The earliest immunoassays developed were of this type • Sensitive • Difficult to automate • Time consuming • Hazardous The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay. Nobel prize in 1977 11 ELISA – Enzyme-linked immunosorbent assay • • • • Method for concentration determination using antibody–based technique Solid-phase assay (polystyrene 96-well plates) Use of enzyme-conjugated secondary antibody Spectrophotometric measurement of the amount of coloured reaction product formed after addition of chromogenic substrate. • Compare result with standard curve measured with known amount of analyte • Different formats available (indirect ELISA, competitive-ELISA and sandwich-ELISA Indirect ELISA; for quantitative determination of antibody concentration against specific antigen. Sandwich-ELISA; for quantitative determination of low concentration antigen in complex solution. Antibody-based home pregnancy test Test for hCG: Human chorionic gonadotropin (pregnancy hormone) Y Y Anti-hCG antibody no. 2 Y Y Y Coloured latex beads + Anti-hCG antibody no. 1 Anti-Anti-hCG antibody Y Test line Control line Capillary force Y Y Y Y or Y Y Y Y Y Y Y Y Y HCG Y Y Y Y Y Positive Negative Invalid 12 Y Western blotting a.k.a. immunoblot • Technique for detection/analysis of either proteins or antibodies in a complex mixture. • The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting. • – Complementary to ELISA techniques, but more informative. Theoretical size of target protein available – – – Can be used to answer questions like: Does my sample contain protein X (if antibodies to X are available)? Does this serum sample contain antibodies to X (if protein x is available)? How specific is my antibody? • • Involves three main steps: 1. SDS-PAGE (electrophoretic size separation of sample in gel) 2. Transfer of size separated sample to nitrocellulose/PVDF-membrane 3. Probing of membrane with antibodies/serum Protein separation by SDS-PAGE Sample loading Sample preparation Marker proteins SS Polyacrylamide gel SDS + Β-mercaptoethanol Native conditions SS - - - SS - - - -- Native molecule Negative charges introduced No 3D-structure Disulphides unaffected Detect proteins - - S H - -- SDS + SH Negative charges introduced No 3D-structure No disulphide bonds No multimers of proteins •Coomassie blue staining •Silver staining 13 Western blotting Transfer proteins to membrane by: •Capillary force •Electrophoresis Sample loading Marker proteins Membrane Polyacrylamide gel + Block membrane and add antibodies, serum etc NB! Epitopes for mAbs can be destroyed by SDS treatment Y Secondary labelled antibody SS Primary antibody YY mAbs recognizing 3D-epitope HIV-test Western blotting • For validation of ELISA results • Interpretation of result is not standardized • In general ”Three band rule” 14 Immunohistochemistry Immunocytochemistry Immunofluorescence Immunoelectron microscopy Immunohistochemistry or IHC refers to the process of localizing proteins in cells of a tissue section Can be used to answer questions like: • In what cells of a particular tissue is a protein expressed? • Where in that cell is the protein localized? Such information is important to elucidate the function of "unknown" proteins. The functions of majority of human proteome are still unknown. TMA – Tissue micro arrays Biobank material TMA production Antibody based tissue profiling in large set of tissues or cells 15 Tissue Micro Array - Production Microtone cut Donor tissue Transfer to recipient block Transfer to glass slide • Protein expression and localization data for normal tissues, cancer tissues, cells and cell lines. • Protein Atlas Ver 3.0 Released in October 8th, 2007 at the HUPO meeting in Seoul, Korea (www.proteinatlas.org) • Publically available database free of charge • Contains >3000 antibodies corresponding to more than 2.8 million annotated immunohistochemical images www.proteinatlas.org 16 Antibody quality assurance 1. Low similarity 2. Protein array 3. Western blot 4. Histochemistry (IHC) Reliability score: High Medium Low Very low Failed Two independent antibodies with similar staining patterns Consistent with bioinformatics and literature (if available) At least some evidence that supports the staining patterns No evidence that the antibody is correct The antibody is not correct TMAs in the Human Protein Atlas project Normal tissues Cancer tissues Cells & cell lines In total 708 cores analyzed per antibody: 48 normal tissues (x 3) 20 cancer tissues (17 x 12 x 2 + 3 x 4 x 2) 58 cell samples (x 2) 17 All validation data available •Gene and protein data (Ensembl, Uniprot) •PrEST data (size, similarity scores) •Antibody info •Validation summary •Protein array validation •Western blot www.proteinatlas.org In silico biomarker discovery Prostate Breast cancer cancer Colon cancer HPA680087 ACPP HPA260028 HPA350021 PSA HPA880064 HPA290101 HPA380031 HPA620019 18 Tissue based search function Advanced tissue based search function in Protein Atlas 3.0 Immunoelectron microscopy Label antibodies with colloidal gold, ferritin or other electron dense labels. Visualized as small black dots with the electron microscope Picture shows surface of a B cell lymphoma stained with two antibodies: -One against MHC Class II, labelled with 30nm gold particles. -One against MHC Class I molecules labelled with 15nm gold particles. (Jenei et al, 1997) Bar = 500nm 19 Immunofluorescence • Novel feature in Protein Atlas 3.0 provide high-resolution multi-color images of immunofluorescently stained cells •Increased spatial information on a fine cellular and subcellular level •Three cell lines, U-2OS, A-431 and U-251MG currently incorporated •Co-staining with cellular probes targeting specific compartments (DAPI, calreticulin, tubulin, HPA-antibody) Flow cytometry • Analysis and sorting of cells • Based on fluorescence and light-scattering properties of individual cells • Cells can be labelled by: labeled antibodies expression of fluorescent reporter • • • • Cells are analyzed on at the time (real-time measurement) Several parameters can be analyzed simultaneously Can be performed at high speed (up to 100.000 events/sec) Cells are often viable after cell sorting 20 The principles of flow cytometry Fluorescence FL3 detection FL2 Side Scatter (complexity) 104 103 FL1 102 101 Forward Light Scatter (object size) Light Source 100 0 200 400 600 800 1000 FSC-H 80 M2 Counts 60 40 20 - 0 + 100 101 102 103 104 FL1-H Data visualisation Sorted target cells Analysis of cell subpopulations 21 Flow cytometry analysis of T-cell populations One healthy and one HIV-infected person FITC T-helper cells (HIV target cells) PE Cytotoxic T-cells Control with labeled but non-specific antibodies Immunoaffinity chromatography Sample loading Washing Elution • Use of antibodies (mAbs or polyclonal) for purification of the antigen • Simultaneous purification and concentration • Antibodies are immobilized onto a chromatographic resin • Stability of the antibody(ies) is important (elution, column sanitation) 22 Choosing the right antibody Every immunochemical technique presents the antigen in a different physical context Often antibodies that work well for a particular technique are a poor choice for another assay Success of an immunochemical technique: •The avidity of antibodies for the antigen •Antibody specificity •Damaged epitopes – How the epitope structure are altered during the technique •Antibody access – how easily the antibody can reach the antigen •Secondary reagents 23