BÀI GIẢNG CHỦ ĐỀ MIỄN DỊCH

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BÀI GIẢNG CHỦ ĐỀ MIỄN DỊCH

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Immunochemical Methods in the Clinical Laboratory Roger L Bertholf, Ph.D., DABCC Chief of Clinical Chemistry & Toxicology, UFHSC/Jacksonville Associate Professor of Pathology, University of Florida College of Medicine Name The Antigen ASCP/Bertholf Early theories of antibody formation • Paul Ehrlich (1854-1915) proposed that antigen combined with pre-existing side-chains on cell surfaces • Ehrlich’s theory was the basis for the “genetic theory” of antibody specificity The “Template” theory of antibody formation • • Karl Landsteiner (1868-1943) was most famous for his discovery of the A/B/O blood groups and the Rh factor Established that antigenic specificity was based on recognition of specific molecular structures; he called these “haptens”; formed the basis for the “template” theory of antibody formation Aminobenzene Sulphonate, a Hapten NH2 NH2 NH2 SO3 SO3 SO3 Ortho Meta Para Classification of immunochemical methods • Particle methods – Precipitation • Immunodiffusion • Immunoelectrophoresis – Light scattering • Nephelometry • Turbidimetry • Label methods – Non-competitive • One-site • Two-site – Competitive • Heterogeneous • Homogeneous Properties of the antibody-antigen bond • Non-covalent • Reversible • Intermolecular forces – Coulombic interactions (hydrogen bonds) – Hydrophobic interactions – van der Waals (London) forces • Clonal variation Antibody affinity Ab + Ag ↔ Ab • Ag [ Ab • Ag ] Ka = [ Ab][ Ag ] Precipitation of antibody/antigen complexes • Detection of the antibody/antigen complex depends on precipitation • No label is involved • Many precipitation methods are qualitative, but there are quantitative applications, too Factors affecting solubility • • • • Size Charge Temperature Solvent ionic strength Substrate-labeled fluorescence immunoassay S Enzyme S No signal Fluorescence S Enzyme Signal Fluorescence excitation transfer immunoassay No signal Signal Electrochemical differential polarographic immunoassay Oxidized Reduced Prosthetic group immunoassay P S Enzyme No signal P P Enzyme Signal Enzyme channeling immunoassay Substrate E1 Product E2 Ag Product Artificial antibodies • Immunoglobulins have a limited shelf life – Always require refrigeration – Denaturation affects affinity, avidity • Can we create more stable “artificial” antibodies? – Molecular recognition molecules – Molecular imprinting History of molecular imprinting • Linus Pauling (1901-1994) first suggested the possibility of artificial antibodies in 1940 • Imparted antigen specificity on native globulin by denaturation and incubation with antigen O - Fundamentals of antigen/antibody interaction Cl O NH + N O- OH NH2 O CH2-CH2-CH2-CH3 Molecular imprinting (Step 1) Methacrylic acid + Porogen O H3C O NH N N CH3 N O H3C O NH N N CH3 N Molecular imprinting (Step 2) O H3C O NH N N CH3 N O H3C O NH N N CH3 N Molecular imprinting (Step 3) Cross-linking monomer Initiating reagent O H3C O NH N N CH3 N O H3C O NH N N CH3 N Molecular imprinting (Step 4) Comparison of MIPs and antibodies Antibodies MIPs • In vivo preparation • In vitro preparation • Limited stability • Unlimited stability • Variable specificity • Predictable specificity • General applicability • Limited applicability Immunoassays using MIPs • Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (α 2adrenoceptor antagonist) • Hormones: Cortisol, Corticosterone • Neuropeptides: Leu5-enkephalin • Other: Atrazine, Methyl-α-glucoside Aptamers 1014-1015 random sequences Target Oligonucleotide-Target complex Unbound oligonucleotides + Target Aptamer candidates PCR New oligonucleotide library ... structures; he called these “haptens”; formed the basis for the “template” theory of antibody formation Aminobenzene Sulphonate, a Hapten NH2 NH2 NH2 SO3 SO3 SO3 Ortho Meta Para Classification of immunochemical

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Mục lục

  • Immunochemical Methods in the Clinical Laboratory

  • Name The Antigen

  • Early theories of antibody formation

  • The “Template” theory of antibody formation

  • Aminobenzene Sulphonate, a Hapten

  • Classification of immunochemical methods

  • Properties of the antibody-antigen bond

  • Antibody affinity

  • Precipitation of antibody/antigen complexes

  • Factors affecting solubility

  • The precipitin reaction

  • Single radial immunodiffusion

  • Slide 13

  • Double immunodiffusion

  • Double immunodiffusion (Ouchterlony)

  • Quantitative double immunodiffusion

  • Electroimmunodiffusion

  • Slide 18

  • Immunoelectrophoresis

  • Slide 20

  • Slide 21

  • Immunofixation electrophoresis

  • Particle methods involving soluble complexes

  • Light reflection

  • Molecular size and scattering

  • Distribution of scattered radiation

  • Nephelometry vs. Turbidimetry

  • Rate nephelometry

  • Additional considerations for quantitative competitive binding immunoassays

  • Competitive immunoassay response curve

  • Logistic equation

  • Logit transformation

  • PowerPoint Presentation

  • High dose “hook” effect

  • Analytical methods using labeled antigens/antibodies

  • Slide 36

  • The birth of immunoassay

  • Radioisotope labels

  • Enzyme labels

  • Fluorescent labels

  • Chemiluminescent labels

  • Slide 42

  • Slide 43

  • Introduction to Heterogeneous Immunoassay

  • Heterogeneous immunoassays

  • Enzyme-linked immunosorbent assay

  • ELISA (variation 1)

  • ELISA (variation 2)

  • Automated heterogeneous immunoassays

  • Immobilized antibody methods

  • Coated tube methods

  • Coated bead methods

  • Microparticle enzyme immunoassay (MEIA)

  • Magnetic separation methods

  • Slide 55

  • Electrochemiluminescence immunoassay (Elecsys™ system)

  • ASCEND (Biosite Triage™)

  • ASCEND

  • Slide 59

  • Solid phase light scattering immunoassay

  • Introduction to Homogeneous Immunoassay

  • Homogeneous immunoassays

  • Typical design of a homogeneous immunoassay

  • Enzyme-multiplied immunoassay technique (EMIT™)

  • EMIT™ method

  • EMIT™ signal/concentration curve

  • Fluorescence polarization immunoassay (FPIA)

  • Molecular electronic energy transitions

  • Polarized radiation

  • Fluorescence polarization

  • Slide 71

  • Fluorescence polarization immunoassay

  • FPIA signal/concentration curve

  • Cloned enzyme donor immunoassay (CEDIA™)

  • Cloned enzyme donor

  • Cloned enzyme donor immunoassay

  • CEDIA™ signal/concentration curve

  • Other approaches to homogeneous immunoassay

  • Substrate-labeled fluorescence immunoassay

  • Fluorescence excitation transfer immunoassay

  • Electrochemical differential polarographic immunoassay

  • Prosthetic group immunoassay

  • Enzyme channeling immunoassay

  • Artificial antibodies

  • History of molecular imprinting

  • Fundamentals of antigen/antibody interaction

  • Molecular imprinting (Step 1)

  • Molecular imprinting (Step 2)

  • Molecular imprinting (Step 3)

  • Molecular imprinting (Step 4)

  • Comparison of MIPs and antibodies

  • Immunoassays using MIPs

  • Aptamers

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