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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 • Research problems requiring immunoanalyses Role of antibodies • Protect against – Viral infections – Bacterial infections – Foreign bodies • Antigens • Deleterious in – Autoimmune diseases • Reumathoid arthritis • Type 1 diabetes – Graft rejection Lupus Croh’n disease 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 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 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) Antigen-antibody binding Structure of an antibody 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 Generation of an antibody: antigen processing B cell activation Antibody and VDJ recombination Generation of antibodies: polyclonal vs monoclonal • Host animals ca be used to raise antibodies against a given antigen • Slected clones from a polyclonal each recognizing a single epitope can be fused to a tumor cell (hybridoma) to proliferate indefinitely 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 Antigen-antibody interaction: concentration dependence Concentration of unknown samples are determined from a standard curve 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.) 90% 10% Doses response curves • Antibody antigen interaction – RIA, ELISA – Ligand receptor interaction – Growth factors – Hormones • Activity of chemotherapeutics • Enzymatic inhibitors Cross reactivity One and two sites competition Detection principles • Radiolabelled isotopes – 125I, 14C, 32P, 35S • Enzymes – Peroxydase • Chromophores – Fluorogenic probes, fluorescent proteins Peroxydase reaction RIA: radio immuno assay RIA interference Elisa: Enzyme-linked immunosorbent assay Sandwich Elisa Western blotting Two dimensional electrophoresis 2nd dimension Molecular weight kDa 1st dimension pH Immunoprecipitation Western Blotting Immunohistochemistry 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) 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) Phosphospecific antibodies to study cellular signaling • 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 DNA damage inducible cascades Phosphospecific detections dsDNA breaks Kinases and signal transduction UV, Inflammator MMS y cytokines Tpl-2 ATM Cdc42 Hs SHPT P1 c-Abl Pyk2 MEKK 1 TAK 1 MEK K4 MAP3Ks MLK s RAF 1 TAO s SEK MK 1 K7 Synergize in SAPK activation SAPK s MK K3 a MK K6 a ATF2 Inhibited by PD98059 (MEK2) ME K5 Inhibited by CSAIDS (CytokineSuppressive AntiInflammatory Drugs) M3/ 6 MK P1 p38 s Pac 1 ERK 5 a MK P5 c-jun MEK K3 Rac1 ASK 1 Ly n MEK K2 NFAT4 , NFAT c1 MAX CHOP/ GADD1 53 MEF2 A-C p53 ELK 1/T CF eg SB203580 CDC2 5B MAPKAPK2/3 MEKs MEK 1/2 MK P2 MK P4 PRAK MK P3 MAPKs ERK1/ Pac (Hematopoi 2 etic 1only) MSK1/2 MNK1/2 Effector Kinases Transcription Factors HSP25/27 WIP PP2B/ CDC2 1 Calcineurin Inhibits nuclear transloca tion RSKs eIF4E CREB, Histone H3, HMG14 Cytoskelet on Translati on Chromatin Remodelli ng FRET: Fluorescence resonance energy transfer 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 cDNA librairies Expression librairies Phage display 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 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) Concentration of serum peptides • Blood levels of: – – – – Hormones Antibodies Enzymes Metabolites 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 The problems of chemotherapy Chemotherapy/ radiotherapy DNA Damage Drug resistance arising from altered drug delivery to target Sensors Drug resistance arising from sensor/transducer defects Transducers Cytoplasmic/Nuclear effectors Chromatin Structure Transcription DNA repair Cell cycle checkpoints Drug resistance arising from effector defects Apoptosis