南京工程学院第三届大学生创业计划 ..

Principles of immunodetection,byMartin Loignon Ph.D.Lady Davis Institute for Cancer ResearchJewish General Hospital,Antibody-based methods allowing the specific: DetectionQuantificationLocalisationOf antigens by means of antibody binding,Immunodetection,Aims and Objectives,Basis of antibody production and antigen interactionConceptualise the different analytical techniques based on this interactionExamples of clinical application Research problems requiring immunoanalysesTroubleshooting of some common problems,Discovery of antibodies,1899 *Jules Bordet, Complement and antibody activity in bacteriolysis 1900 *Paul Erlich, Antibody formation theory 1926 Lloyd Felton & GH Bailey, Isolation of pure antibody preparation 1934-8 John Marrack, Antigen-antibody binding hypothesis 1941 Albert Coons, Immunofluorescence technique 1948 Astrid Fagraeus, Demonstration of antibody production in plasma B cells 1959-62 *Rodney Porter et al., Discovery of antibody structure 1963 Jaques Oudin et al., antibody idiotypes 1964-8 Anthony Davis et al., T and B cell cooperation in immune response 1965 Thomas Tomasi et al., Secretory immunoglobulin antibodies 1975 *Kohler and Milstein, Monoclonal antibodies used in genetic analysis 1985 *Tonegawa, Hood et al., Identification of immunoglobulin genes,Generation of an antibody: antigen processing,B cell activation,Structure of an antibody,Antibody and VDJ recombination,Classes of antibodies,Commercial production 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,Antigen-antibody interaction,Antigen: foreign molecules that generate antibodies or any substance that can be bound specifically by an antibody moleculeProteins, sugars, lipids or nucleic acidsNatural or synthetic Antibody: molecules (protein) responsible for specific recognition and elimination (neutralization) of antigensDifferent structures (7-8 classes in mammals)Powefull research tools for basic research, clinical applications and drug design,Antigenic determinants,An antibody will recognizeEpitope: defined segment of an antigenImmunoreactivity of epitopes may depend on primary, secondary, tertiary or quaternary structure of an antigenDefine the possible applicationsVariability of epitopes depends on the speciesAntibodies are antigen themselves,Nature of binding forces,Hydrogen bondingResults from the formation of hydrogen bridges between appropriate atomsElectrostatic forcesAre due to the attraction of oppositely charged groups located on two protein side chainsVan 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,Antigen-antibody affinity,The affinity with which antibody binds antigen results from a balance between the attractive and repulsive forces. A high affinity antibody impliesa good fit and conversely, a low affinity antibody implies a poor fit and a lower affinity constant,Antigen-antibody interaction: concentration dependence,Concentration of unknown samples are determined from a standard curveSTD concentration values are obtained when the interaction between,Non specific binding,Saturation radioligand binding experiments measure specific radioligand binding at equilibrium at various concentrations of the radioligand. These experiments are performed to determine receptor number and affinity on cells but also between radiolabeled antigen and Ab. This can take anywhere from a few minutes to many hours, depending on the ligand, receptor, To, and other experimental conditions.The lowest concentration of radioligand will take the longest to equilibrate. When testing equilibration time, therefore, use a low concentration of radioligand (perhaps 10-20% of the KD). Nonspecific binding is almost always a linear function of ligand concentration.The analyses depend on the assumption that you have allowed the incubation to proceed to equilibrium.,Dissociation off rate experiments,Each ligand-receptor complex dissociates at a random time, so the amount of specific binding follows an exponential dissociation.,General equation for a dose response curveIt shows response as a function of the logarithm of concentrationX is the logarithm of agonist concentration and Y is the responseLog EC50 is the logarithm of the EC50 (effective concentration, 50% of maximal response)IC50 (inhibitory conc.),Sigmoidal dose response curve,Ligand receptor interactionGrowth factorsHormonesAntibody antigen interaction RIA, ELISAActivity of chemotherapeuticsEnzymatic activators/inhibitors,Doses response curves,Cross reactivity,One and two sites competition,Laboratory use of antibodies,Quantitation of an antigenRIA, ElisaIdentification and characterization of protein antigensImmunoprecipitation Western blottingCell surface labelling and separationLocalisation of antigens within tissues or cellsExpression librairiesPhage display,Detection principles,Radiolabelled isotopes (antigen)125I, 32P, 35SEnzymes (Ab)PeroxydaseChromophores (Ab)Fluorogenic probes (UV, visible or IR),Peroxydase reaction,RIA: radio immuno assay,Typical RIA standard curve,RIA interference,Elisa: Enzyme-linked immunosorbent assay,Sandwich Elisa,Western blotting,Two dimensional electrophoresis,pH,Molecular weight kDa,1st dimension,2nd dimension,Stable pH gradient,Immunoprecipitation,ProteomicsWestern Blotting,Immunohistochemistry,Phosphorylation and dephosphorylation affect the structure and activity of proteinsCellular signalling is characterized by cascades of phosphorylationKinases and phosphatases maintain phosphorylated/dephosphorylated state of proteins Phospho/Tyrosine/Threonine/ Serine,Phosphospecific antibodies to study cellular signaling,DNA damage inducible cascades,Phosphospecific detections,Phospho Ser, Thr, TyrSequence specific (a-Ser18 p53),Antibodies against other post- translational modifications,UbiquitinationSumoylationAcetylationMethylationGeranylationEtc.,Specific DNA damage (CPD, 6-4PP)SugarsLipidsVitamins (vit D)Iodine,Antibodies against non-protein antigens,Identification of signaling pathwaysProtein modificationsSignaling partnersActivity of drugs (lead compounds)Lack of specific moleculesSpecific ligands (side effects)New antibodies,Research requiring immunoanalyses,Kinases and signal transduction,Phage display,Bacteriophage structure,Production of recombinant phages,cDNA librairies,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,Alternatives to specific antibodies,Gene of interest,Fluoresentproteins,CFP,GFP,YFP,RFP,a-FP Ab,Direct visualisation,TAGS,His,Myc,Flag,Strep,GST,Affinity,a-Tag Ab,FRET: Fluorescence resonance energy transfer,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,Localization of CEBP by FRET,Clinical use of antibodies,DiagnosticDetection of peptides and other molecules in various diseasesEndocrine diseases: hyperinsulinemia, diabetes, hyperparatyroidismTumor antigens (p53 tumor suppressor, PSA, a-foetoprotein)Antibodies against viral proteins (AIDS, hepatitis)Therapeutic Neutralizing antibodiesAnti-ErbB2 for breast and ovarian cancerAnti-CD20 for B-cell non-Hodgkins lymphomaAntisera and antidotes (viruses and venoms)Drug discoveryIdentification of therapeutic targets (phage display),Therapeutic applications,Neutralizing antibodiesAntidotes and antivenin (snake & spider bites)Tumor antigens ErbB-2, melanoma and T-cell leukemia, antibodies coupled to toxinsAutoimmune 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 antibodiesComplement activating for uncontrolled bleeding (hemophilia),Concentration of serum peptides,Blood levels of:HormonesAntibodiesEnzymesMetabolites,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),Immunodiffusion,Zone of equivalence:formation of large complexes,The problems of chemotherapy,Chemotherapy/radiotherapy,Sensors,Transducers,Cytoplasmic/Nuclear effectors,Chromatin StructureTranscription,DNA repairCell cycle checkpoints,Apoptosis,Drug resistance arisingfrom sensor/transduce