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Principles of immunodetection

Principles of immunodetection . by Martin Loignon Ph.D. Lady Davis Institute for Cancer Research Jewish General Hospital. Aims and Objectives. Basis of antigen-antibody interaction Conceptualise the different techniques based on this interaction Examples of clinical application

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Principles of immunodetection

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  1. Principles of immunodetection by Martin Loignon Ph.D. Lady Davis Institute for Cancer Research Jewish General Hospital

  2. Aims and Objectives • Basis of antigen-antibody interaction • Conceptualise the different techniques based on this interaction • Examples of clinical application • Research problems requiring immunoanalyses

  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. Antigen-antibody interaction • Antigen: foreign molecules that generate antibodies or any substance that can be bound specifically by an antibody molecule • Proteins, sugars, lipids or nucleic acids • Natural or synthetic • Antibody: molecules (protein) responsible for specific recognition and elimination (neutralization) of antigens • Different structures (7-8 classes in mammals) • Powefull research tools for basic research, clinical applications and drug design

  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. 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 side chains • 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 water molecules is minimized (may contribute up to half the total strength of the antigen-antibody bond)

  7. Antigen-antibody binding

  8. Structure of an antibody

  9. Antigen-antibody affinity The affinity with which antibody binds antigen results from a balance between the attractive and repulsive forces. A high affinity antibody implies a good fit and conversely, a low affinity antibody implies a poor fit and a lower affinity constant

  10. Generation of an antibody: antigen processing

  11. B cell activation

  12. Antibody and VDJ recombination

  13. Host animals ca be used to raise antibodies against a given antigen Generation of antibodies: polyclonal vs monoclonal • Slected clones from a polyclonal each recognizing a single epitope can be fused to a tumor cell (hybridoma) to proliferate indefinitely

  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. Antigen-antibody interaction: concentration dependence Concentration of unknown samples are determined from a standard curve

  16. 90% 10% Sigmoidal dose response curve • General equation for a dose response curve • It shows response as a function of the logarithm of concentration • X is the logarithm of agonist concentration and Y is the response • Log EC50 is the logarithm of the EC50 (effective concentration, 50%) • IC50 (inhibitory conc.)

  17. Doses response curves • Antibody antigen interaction • RIA, ELISA • Ligand receptor interaction • Growth factors • Hormones • Activity of chemotherapeutics • Enzymatic inhibitors

  18. Cross reactivity

  19. One and two sites competition

  20. Detection principles • Radiolabelled isotopes • 125I, 14C, 32P, 35S • Enzymes • Peroxydase • Chromophores • Fluorogenic probes, fluorescent proteins

  21. Peroxydase reaction

  22. RIA: radio immuno assay

  23. RIA interference

  24. Elisa: Enzyme-linked immunosorbent assay

  25. Sandwich Elisa

  26. Western blotting

  27. Two dimensional electrophoresis 1st dimension 2nd dimension Molecular weight kDa pH

  28. Immunoprecipitation Western Blotting

  29. Immunohistochemistry

  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-Hodgkin's lymphoma • Experimental • Drug screening (phage display)

  31. Detection of HIV proteins by WB gp160 viral envelope precursor (env) gp120 viral envelope protein (env) binds to CD4 p31 Reverse Transcriptase (pol) p24 viral core protein (gag)

  32. Phosphorylation and dephosphorylation affect the structure and activity of proteins Cellular signalling is characterized by cascades of phosphorylation Kinases and phosphatases maintain phosphorylated/dephosphorylated state of proteins Phospho/Tyrosine/Threonine/ Serine Phosphospecific antibodies to study cellular signaling

  33. DNA damage inducible cascades

  34. Phosphospecific detections

  35. dsDNA breaks UV, MMS Inflammatory cytokines Tpl-2 MEKK2 MEKK3 c-Abl Cdc42Hs Rac1 ATM SHPTP1 ASK1 TAK1 MEKK4 Pyk2 Lyn MAP3Ks RAF1 MEKK1 MLKs TAOs Inhibited by PD98059 (MEK2) MEKs SEK1 MKK7 MKK3 MKK6 MEK5 MEK1/2 Inhibited by CSAIDS (Cytokine-Suppressive Anti-Inflammatory Drugs) eg SB203580 Synergize in SAPK activation   MAPKs Pac1 ERK1/2 (Hematopoietic only) SAPKs p38s ERK5  CDC25B MAPKAP-K2/3 PRAK MSK1/2 MNK1/2 RSKs c-jun ATF2 NFAT4, NFAT c1 MAX CHOP/ GADD153 MEF2A-C p53 ELK1/TCF Effector Kinases Transcription Factors HSP25/27 CREB, Histone H3, HMG14 CDC2 eIF4E MKP2 MKP1 MKP3 M3/6 WIP1 Pac1 MKP5 MKP4 Inhibits nuclear translocation PP2B/ Calcineurin Cytoskeleton Chromatin Remodelling Translation Kinases and signal transduction

  36. FRET: Fluorescence resonance energy transfer

  37. Localization of CEBP by FRET Localization of BFP- and RFP-C/EBP protein expressed in mouse 3T3 cells using 2p-FRET microscopy. The doubly expressed cells (BFP-RFP-C/EBP) were excited by 740 nm and the donor (A) and acceptor (B) images of proteins localized in the nucleus of a single living cell were acquired by single scan

  38. cDNA librairies

  39. Expression librairies

  40. Phage display

  41. Phage display: Ab production Originally developped to produce monoclonal antibodies, phage display is a simple yet powerful technology that is used to rapidly characterize protein-protein interactions from amongst billions of candidates. This widely practiced technique is used to map antibody epitopes, create vaccines and to engineer peptides, antibodies and other proteins as both diagnostic tools and as human therapeutics

  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. Concentration of serum peptides • Blood levels of: • Hormones • Antibodies • Enzymes • Metabolites

  44. Research problems requiring immunoanalyses • Identification of signaling pathways • Protein modifications • Signaling partners • Activity of drugs (lead compounds) • Lack of specific molecules • Specific ligands (side effects) • New antibodies

  45. The problems of chemotherapy Chemotherapy/ radiotherapy Drug resistance arising from altered drug delivery to target DNA Damage Sensors Drug resistance arising from sensor/transducer defects Transducers Drug resistance arising from effector defects Cytoplasmic/Nuclear effectors • DNA repair • Cell cycle • checkpoints • Chromatin • Structure • Transcription • Apoptosis

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