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8th SEMINAR

8th SEMINAR. LABORATORY METHODS BASED ON ANTIGEN-ANTIBODY INTERACTIONS I. THE SENSITIVITY OF IMMUNOASSAYS. S ensitive methods : precise expensive usually used for verification. Less sensitive methods : give semiquantitative results cheap usually used for screening.

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8th SEMINAR

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  1. 8th SEMINAR LABORATORY METHODS BASED ON ANTIGEN-ANTIBODY INTERACTIONS I

  2. THE SENSITIVITY OF IMMUNOASSAYS • Sensitivemethods: • precise • expensive • usuallyusedforverification • Less sensitivemethods: • givesemiquantitativeresults • cheap • usuallyusedforscreening

  3. IMMUNOAFFINITY CHROMATOGRAPHY Separation/purification of antigens orantibodies from a mixture

  4. AFFINITY PURIFICATION OF ANTIBODIES USING AN ANTIGEN-SORBENT COLUMN column polymer beads affinity purified antibody : monoclonal antibodies which can be ordered from catalogues are also purified using this technique covalently bound antigen

  5. STEPS OF PURIFICATION • Addition of antibodies to be purified • Binding • Washing • Elution

  6. column fixed antigen-specific Abs on the surface of the bead PURIFICATION OF ANTIGENS • Loading the antigen mixture • Binding • Washing • Elution polymer bead Purified antigens

  7. ELISAEnzyme Linked Immune Sorbent Assay ELISA plate well

  8. enzyme Immune Sorbent Enzyme Linked Antigen/antibody adsorbed to solid surface Antibody conjugated with enzyme

  9. 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

  10. Label Secondary antibodies Label Antigen BASIC SETUPS INELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Direct method Indirect method Primary antibodies

  11. Enzyme Enzyme-specific antibody, same isotype as the primary antibody Secondary antibody Primary antibody Antigen BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Enzyme/anti-enzyme system PAP – peroxidase / anti-peroxidase APAAP – alkaline phosphatase / anti- alkaline phosphatase

  12. Avidin-biotin enzyme complexes Avidin-enzymecomplexes Biotin-enzyme complex Avidin Biotinylated antibody Antigen BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity ) ABC Basic

  13. STEPS OF COMBINED/’SANDWICH’ ELISA For antigens present at low concentration in complex biological samples Coating with Ag-specific „capture” antibody Removal of excess enzyme Removal of unbound material Blocking free plastic surface with inert protein Removal of unbound protein Removal of unbound material Addition of antigen- containing solution Addition of biotinylated antibody specific to a different epitope on target protein Addition of avidin-conjugated enzyme Addition of substrate

  14. 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 Removal of excess antibody Saturation of uncovered surface area with proteins Addition of Ag-specific antibodies Addition of Secondary Ab conjugated with enzyme Addition of chromogenic substrate

  15. 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 ? 62 31 16 7.8 3.9 1.9 500 250 125 0 0.97 0.49 0.24 0.12 1000 0.061 0.030 0.015 0.007 0.004 concentration

  16. PRACTICAL USE OF ELISA TECHNICS Sandwich ELISA Indirect ELISA Measuring the amount of antigen-specific antibodies pathogen-specific antibodies – diagnosis of infection* isotype of antibodies – time course, monoclonal antibodies autoantibodies – diagnosis of autoimmune disorders Measuring the amount of a given antigen (molecule) • cytokines, hormones, drugs • viral/bacterial antigens – diagnosis of infection • tumor antigens – diagnosis of tumors / screening / follow-up

  17. WESTERN BLOT (IMMUNOBLOT) Identification of defined components from protein mixtures by antigen specific antibodies Anode(+) Steps: • Sample preparation (cells, tissues) • Gel electrophoresis • Blotting • Labeling (by primary and secondary antibodies) • Detection Cathode(-)

  18. WESTERN BLOT (IMMUNOBLOT) Lysis of sample Loading Gel-electrophoresis Blotting Labeling Primary antibody binds to its epitope in the protein, then the labeled secondary antibody binds to the primary antibody Standard Protein sample SDS-PAGE Membrane X-ray film

  19. IMMUNOPRECIPITATION Isolation and concentration of a particular protein from a protein mixture Detectionof protein associations (e.g. members of receptor signalization)

  20. 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

  21. 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)

  22. IMMUNOHISTOCHEMISTRY Fixation Sectioning Tissue sample Section before staining Freezing

  23. IMMUNOHISTOCHEMISTRY Enzyme Avidin Secondary antibody X Biotin Primary antibody Slide Cells Tissue sample

  24. Classical histochemistry Acute bronchopneumonia (hematoxylin-eozin staining) Only few cell types could be identified

  25. Immunohistochemistry (CD68+ macrophages and lymphocytes, granuloma)

  26. Antinuclear (ANA) autoantibodies from the serum of a SLE patient can be visualized in cell culture (HEp-2) by indirect fluorescent labeling (immunofluorescence)

  27. A fixed and permeabilized skin fibroblast Mitochondria F-actin Nucleus Fixed and permeabilized pulmonary artery endothelial cell Peroxisomes MitochondriaNuclei

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