Transcript 4th SEMINAR
8th SEMINAR LABORATORY METHODS BASED ON ANTIGEN-ANTIBODY INTERACTIONS I THE SENSITIVITY OF IMMUNOASSAYS Sensitive methods: • precise • expensive • usually used for verification Less sensitive methods: • give semiquantitative results • cheap • usually used for screening IMMUNOAFFINITY CHROMATOGRAPHY Separation/purification of antigens or antibodies from a mixture AFFINITY PURIFICATION OF ANTIBODIES USING AN ANTIGEN-SORBENT COLUMN column affinity purified antibody : monoclonal antibodies which can be ordered from catalogues are also purified using this technique polymer beads covalently bound antigen STEPS OF PURIFICATION 1) 2) 3) 4) Addition of antibodies to be purified Binding Washing Elution PURIFICATION OF ANTIGENS 1) Loading the antigen mixture 2) Binding column 3) Washing 4) Elution polymer bead fixed antigen-specific Abs on the surface of the bead Purified antigens ELISA Enzyme Linked Immune Sorbent Assay ELISA plate well Enzyme Linked Immune Sorbent enzyme Antibody conjugated with enzyme Antigen/antibody adsorbed to solid surface ENZYME ACTIVITY IN ELISA IS DIRECTLY PROPORTIONAL TO THE AMOUNT OF IMMUNECOMPLEX PRESENT Enzyme activity is measured by the color reaction due to conversion of substrate Similar principle applies to many other antibody-based detection methods BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Direct method Indirect method Label Label Secondary antibodies Primary antibodies Antigen BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Enzyme/anti-enzyme system PAP – peroxidase / anti-peroxidase APAAP – alkaline phosphatase / anti- alkaline phosphatase Enzyme Enzyme-specific antibody, same isotype as the primary antibody Primary antibody Antigen Secondary antibody BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity ) Basic Avidin-enzyme complexes ABC Avidin-biotin enzyme complexes Biotin-enzyme complex Avidin Biotinylated antibody Antigen STEPS OF COMBINED/’SANDWICH’ ELISA For antigens present at low concentration in complex biological samples Coating with Ag-specific „capture” antibody Blocking free plastic surface with inert protein Removal of excess enzyme Removal of unbound material Removal of unbound protein Removal of unbound material Addition of antigencontaining solution Addition of biotinylated antibody specific to a different epitope on target protein Addition of avidinconjugated enzyme Addition of substrate STEPS OF BASIC INDIRECT ELISA Detection of antigen or specific antibody Removal of excess antigen Removal of excess protein Adsorption of antigen (coating) Removal of excess antibody Saturation of uncovered surface area with proteins Removal of excess antibody Addition of Agspecific antibodies Addition of Secondary Ab conjugated with enzyme Addition of chromogenic substrate EQUAL ABSORBANCE = EQUAL CONCENTRATION OD You should also dilute the unknown sample The sample with unknown concentration This region could indicate the concentration According to OD: it could be anyone concentration 0.004 0.007 0.015 0.030 0.061 0.12 0.24 0.49 0.97 1.9 3.9 7.8 16 31 62 125 250 500 1000 ? 0 PRACTICAL USE OF ELISA TECHNICS Sandwich ELISA Indirect ELISA Measuring the amount of a given antigen (molecule) Measuring the amount of antigenspecific antibodies • cytokines, hormones, drugs • viral/bacterial antigens – diagnosis of infection • tumor antigens – diagnosis of tumors / screening / follow-up • pathogen-specific antibodies – diagnosis of infection* • isotype of antibodies – time course, monoclonal antibodies • autoantibodies – diagnosis of autoimmune disorders WESTERN BLOT (IMMUNOBLOT) Identification of defined components from protein mixtures by antigen specific antibodies Anode(+) Steps: 1) Sample preparation (cells, tissues) 2) Gel electrophoresis 3) Blotting 4) Labeling (by primary and secondary antibodies) 5) Detection Cathode(-) WESTERN BLOT (IMMUNOBLOT) Lysis of sample Loading Gel-electrophoresis Standard Protein sample SDS-PAGE Blotting Labeling Primary antibody binds to its epitope in the protein, then the labeled secondary antibody binds to the primary antibody Membrane X-ray film IMMUNOPRECIPITATION • Isolation and concentration of a particular protein from a protein mixture • Detection of protein associations (e.g. members of receptor signalization) CHROMATIN IMMUNOPRECIPITATION (ChIP) Identification of molecules (mainly transcription factors) binding to a specific site of the DNA Provides information about the link between signaling pathways and gene activation IMMUNOHISTOCHEMISTRY Labeled antibodies added to fixed tissue sections detect the distribution of the chosen antigen within the tissue or within the cells of a particular tissue • Immunofluorescence •Fluorescent dye coupled to antibody FITC – fluorescein isothiocyanate (green) PE – phycoerythrin (orange) • Immunoenzyme method • enzyme-coupled antibody P – peroxidase AP – alkaline phosphatase (Substrates converted into an insoluble compound) IMMUNOHISTOCHEMISTRY Fixation Tissue sample Freezing Sectioning Section before staining IMMUNOHISTOCHEMISTRY Enzyme Secondary antibody Primary antibody X Avidin Biotin Slide Cells Tissue sample Classical histochemistry Acute bronchopneumonia (hematoxylin-eozin staining) Only few cell types could be identified Immunohistochemistry (CD68+ macrophages and lymphocytes, granuloma) Antinuclear (ANA) autoantibodies from the serum of a SLE patient can be visualized in cell culture (HEp-2) by indirect fluorescent labeling (immunofluorescence) A fixed and permeabilized skin fibroblast Mitochondria F-actin Nucleus Fixed and permeabilized pulmonary artery endothelial cell Peroxisomes Mitochondria Nuclei