Principles of immunodetection


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Principles of immunodetection

  1. 1. Principles ofimmunodetection
  2. 2. Aims and Objectives• Basis of antigen-antibody interaction• Conceptualise the different techniquesbased on this interaction• Examples of clinical application• Research problems requiringimmunoanalyses
  3. 3. Role of antibodies• Protect against– Viral infections– Bacterial infections– Foreign bodies• Antigens• Deleterious in– Autoimmune diseases• Reumathoid arthritis Lupus• Type 1 diabetes Croh’n disease– Graft rejection
  4. 4. Antigen-antibody interaction• Antigen: foreign molecules that generate antibodies or anysubstance that can be bound specifically by an antibodymolecule– Proteins, sugars, lipids or nucleic acids– Natural or synthetic• Antibody: molecules (protein) responsible for specificrecognition and elimination (neutralization) of antigens– Different structures (7-8 classes in mammals)– Powefull research tools for basic research, clinical applications anddrug design
  5. 5. Antigenic determinants• An antibody will recognize– Epitope: defined segment of an antigen– Immunoreactivity of epitopes may depend on primary,secondary, tertiary or quaternary structure of an antigen– Variability of epitopes depends on the species• Antibodies are antigen themselves
  6. 6. Nature of binding forces• Hydrogen bonding– Results from the formation of hydrogen bridges between appropriate atoms• Electrostatic forces– Are due to the attraction of oppositely charged groups located on two protein sidechains• Van der Waals bonds– Are generated by the interaction between electron clouds (oscillating dipoles)• Hydrophobic bonds– Rely upon the association of non-polar, hydrophobic groups so that contact with watermolecules is minimized (may contribute up to half the total strength of the antigen-antibodybond)
  7. 7. Antigen-antibody binding
  8. 8. Structure of an antibody
  9. 9. Antigen-antibody affinityThe affinity with which antibody binds antigen results from a balancebetween the attractive and repulsive forces. A high affinity antibody impliesa good fit and conversely, a low affinity antibody implies a poor fit and alower affinity constant
  10. 10. Generation of an antibody:antigen processing
  11. 11. B cell activation
  12. 12. Antibody and VDJ recombination
  13. 13. Generation of antibodies:polyclonal vs monoclonal• Host animals ca be used to raise antibodiesagainst a given antigen• Slected clones from a polyclonal each recognizinga single epitope can be fused to a tumor cell(hybridoma) to proliferate indefinitely
  14. 14. Laboratory use of antibodies• Quantitation of an antigen– RIA, Elisa• Identification and characterization of protein antigens– Immunoprecipitation– Western blotting• Cell surface labelling and separation• Localisation of antigens within tissues or cells• Expression librairies• Phage display
  15. 15. Antigen-antibody interaction:concentration dependenceConcentration of unknown samples are determined from a standard curve
  16. 16. • General equation for adose response curve• It shows response as afunction of the logarithmof concentration• X is the logarithm ofagonist concentrationand Y is the response• Log EC50 is thelogarithm of the EC50(effective concentration,50%)• IC50 (inhibitory conc.)Sigmoidal dose response curve10%90%
  17. 17. • Antibody antigen interaction– RIA, ELISA– Ligand receptor interaction– Growth factors– Hormones• Activity of chemotherapeutics• Enzymatic inhibitorsDoses response curves
  18. 18. Cross reactivity
  19. 19. One and two sites competition
  20. 20. Detection principles• Radiolabelled isotopes– 125I, 14C, 32P, 35S• Enzymes– Peroxydase• Chromophores– Fluorogenic probes, fluorescent proteins
  21. 21. Peroxydase reaction
  22. 22. RIA: radio immuno assay
  23. 23. RIA interference
  24. 24. Elisa: Enzyme-linked immunosorbent assay
  25. 25. Sandwich Elisa
  26. 26. Western blotting
  27. 27. Two dimensional electrophoresispHMolecularweightkDa1st dimension 2nd dimension
  28. 28. ImmunoprecipitationWestern Blotting
  29. 29. Immunohistochemistry
  30. 30. Clinical use of antibodies• Diagnostic– Detection of peptides and other molecules in various diseases• Endocrine diseases: hyperinsulinemia, diabetes, hyperparatyroidism• Tumor antigens (p53 tumor suppressor, PSA, a-foetoprotein)• Antibodies against viral proteins (AIDS, hepatitis)• Therapeutic– Neutralizing antibodies• Anti-Erbb2 for breast and ovarian cancer• Anti-CD20 for B-cell non-Hodgkins lymphoma• Experimental– Drug screening (phage display)
  31. 31. Detection of HIV proteins by WBgp160 viral envelope precursor (env)gp120 viral envelope protein (env) binds to CD4p31 Reverse Transcriptase (pol)p24 viral core protein (gag)
  32. 32. • Phosphorylation and dephosphorylation affectthe structure and activity of proteins• Cellular signalling is characterized by cascadesof phosphorylation• Kinases and phosphatases maintainphosphorylated/dephosphorylated state ofproteins• Phospho/Tyrosine/Threonine/ SerinePhosphospecific antibodies to studycellular signaling
  33. 33. DNA damage inducible cascades
  34. 34. Phosphospecific detections
  35. 35. Cytoskeleton TranslationERK5ELK1/TCFMEF2A-CATF2 NFAT4, NFATc1MAX CHOP/GADD153Transcription Factorsc-junSAPKsInhibitsnucleartranslocationEffectorKinasesMAPKAP-K2/3PRAK MSK1/2 MNK1/2 RSKsp38sHSP25/27CREB, HistoneH3, HMG14eIF4EChromatinRemodellingASK1Tpl-2 MEKK2MEKK3MEKK1RAF1SEK1MKK7aMKK3aMKK6MEK5MEK1/2ERK1/2MAP3KsMAPKsMEKsaInhibited byCSAIDS(Cytokine-SuppressiveAnti-InflammatoryDrugs)eg SB203580Synergize inSAPKactivationp53PP2B/CalcineurinMKP1CDC25BCDC2WIP1Pac1Pac1MKP5MKP4MKP2MKP3M3/6(Hematopoietic only)Inhibited byPD98059(MEK2)c-AblRac1dsDNAbreaksInflammatory cytokinesATMMEKK4TAK1TAOsMLKsUV,MMSPyk2 LynSHPTP1Cdc42HsKinases and signal transduction
  36. 36. FRET:Fluorescence resonance energy transfer
  37. 37. Localization of BFP- and RFP-C/EBP protein expressed in mouse 3T3 cells using2p-FRET microscopy. The doubly expressed cells (BFP-RFP-C/EBP) were excitedby 740 nm and the donor (A) and acceptor (B) images of proteins localized in thenucleus of a single living cell were acquired by single scanLocalization of CEBP by FRET
  38. 38. cDNA librairies
  39. 39. Expression librairies
  40. 40. Phage display
  41. 41. Phage display: Ab productionOriginally developped to produce monoclonalantibodies, phage display is a simple yetpowerful technology that is used to rapidlycharacterize protein-protein interactions fromamongst billions of candidates. This widelypracticed technique is used to map antibodyepitopes, create vaccines and to engineerpeptides, antibodies and other proteins as bothdiagnostic tools and as human therapeutics
  42. 42. Clinical applications• Neutralizing antibodies– Antidotes and antivenin (snake & spider bites)– Tumor antigens ErbB-2, melanoma and T-cell leukemia,antibodies coupled to toxins– Autoimmune antibodies, cytokines TNF-a– Antisera aigainst virus, bateria and toxins (vaccine)– Anti IgE and IgM for allegies (experimental)– Quantitation of blood peptides (hormones metabolites)• Activating antibodies– Complement activating for uncontrolled bleeding (hemophilia)
  43. 43. Concentration of serum peptides• Blood levels of:– Hormones– Antibodies– Enzymes– Metabolites
  44. 44. • Identification of signaling pathways– Protein modifications– Signaling partners• Activity of drugs (lead compounds)• Lack of specific molecules– Specific ligands (side effects)– New antibodiesResearch problems requiringimmunoanalyses
  45. 45. The problems of chemotherapyChemotherapy/radiotherapySensorsTransducersCytoplasmic/Nuclear effectorsChromatinStructureTranscriptionDNA repairCell cyclecheckpointsApoptosisDrug resistance arisingfrom sensor/transducerdefectsDrug resistance arisingfrom effector defectsDNA DamageDrug resistance arisingfrom altered drugdelivery to target