Immunology Lecture Slides - BMS 545 (PDF)

WELCOME- BMS 545 IMMUNOLOGY NOVEMBER 4, 2024   Office hours:  Tues. 4-5 pm (virtual) ANNOUNCEMENTS  Thurs. 4-5 pm (in-person)  Exam Friday 11/8 2:30 pm  THE REST OF THE SEMESTER (ALL MODULE 4)  11/4- Immunological techniques (Exam 4) ...

WELCOME- BMS 545 IMMUNOLOGY NOVEMBER 4, 2024   Office hours:  Tues. 4-5 pm (virtual) ANNOUNCEMENTS  Thurs. 4-5 pm (in-person)  Exam Friday 11/8 2:30 pm  THE REST OF THE SEMESTER (ALL MODULE 4)  11/4- Immunological techniques (Exam 4)  11/6- Vaccines (Exam 4)  11/8- Exam 3 2:30 pm  11/11- PBS Vaccine documentary (Asynchronous)  11/13- Primary Immunodeficiency  11/15- Secondary Immunodeficiency (Asynchronous). Research Days 1-4 pm  11/18- Transplantation & Immune Pharmacotherapy  11/20- Transplantation & Immune Pharmacotherapy & Cancer  11/22- Cancer & Tumor Immunity  11/25- Hypersensitivity (Asynchronous- Enjoy Thanksgiving break)  11/27-11/29- Thanksgiving break  12/2- Autoimmune Disorders  12/4- “What’s Wrong with Me?” Case Study  12/6- Final Immunology debrief: The Well Patient  LEARNING OBJECTIVES  Compare & contrast sensitivity & specificity (& be able to apply it)  Understand antibody-antigen interactions, especially how the equation relates to affinity  Describe the precipitin reaction, the quantitative curve, and the three zones  Define the different laboratory tests used in immunology and describe the protocols and the circumstances in which they would be used  Interpret test results and be able to define when we would need a qualitative vs. quantitative test (and which test we could use to answer the question) DIAGNOSTIC SENSITIVITY AND SPECIFICITY: HOW RELIABLE IS THE TEST?  Interpretation of test results requires an understanding of the test’s reliability prior to diagnosis or treatment  No test is perfect; every testing method produces several false-positive & false-negative results  How much emphasis should be placed on a particular test depends on sensitivity & specificity  Sensitivity- probability that the test will be positive in a patient who has the disease in question  Specificity- probability that the result will be negative in a patient who does not have the disease  Sensitivity & specificity of a test can be determined by using a 2 × 2 table (a, b, c, & d are actual numbers of observations, not proportions) TRUE VS FALSE  A true positive is an outcome where the model correctly predicts the positive outcome  A true negative is an outcome where the model correctly predicts the negative outcome  A false positive is an outcome where the model incorrectly predicts the positive outcome  A false negative is an outcome where the model incorrectly predicts the negative outcome  LET’S APPLY IT!  Imagine the evaluation of a diagnostic test developed to predict the dreaded disease Examinus paralysis, commonly known as “brain freeze,” among students about to take an exam. Data from previous experience indicates that: True positive True negative -------------------------- -------------------------- True pos + false neg False pos + true neg WHAT IS THE SENSITIVITY? WHAT IS THE SPECIFICITY  ANTIGEN-ANTIBODY REACTIONS  Antigen (Ag)–antibody (Ab) interactions are some of the most specific noncovalent biochemical reactions known and can be represented by:  Although the reaction is driven to the right, favoring binding and formation of Ag–Ab complexes, the process is reversible  Affinity- Strength of interaction (i.e.,Ag–Ab association, right arrow, over the dissociation of Ag from Ab, left arrow)  Different immunoglobulins within an individual show a wide range of affinity  Valence- # of epitope-binding sites on an immunoglobulin molecule and varies from two (monomeric forms of all isotypes) to four (secretory IgA) to ten (for pentameric IgM)  Avidity- collective affinity of multiple binding sites (affinity + valence) of an immunoglobulin  PRECIPITIN REACTION  Precipitin reaction- the interaction of soluble antigen with soluble antibody that results in formation of Ag–Ab complexes (lattices) large enough to precipitate from solution  First quantitative assay for antibody  Quantitative precipitin curve- mixing & incubating varying amounts of antigen (in constant volume) with equal & constant volumes of antiserum (containing antibodies) *similar curve can be generated by keeping antigen constant & varying amount of antibody added  Precipitate formation in a series of tubes can be measured & used to describe 3 zones of the quantitative precipitin curve: 1. Zone of Ag Excess 2. Equivalence 3. Zone of Ab Excess  Amount of precipitate formed depends on ratio of antigen to antibody & is also affected by antibody’s avidity  PRECIPITIN REACTION  The three zones of the quantitative precipitin curve: 1. Zone of Ag excess: insufficient antibody to form large lattices. & antigen–antibody complexes are too small to precipitate leading to formation of soluble complexes. 2. Equivalence zone: Optimal precipitation occurs here. Large lattices can be formed, and visible precipitating complexes are formed. 3. Zone of Ab excess: Not enough antigen is present to form large lattices, leading to formation of soluble complexes.  These principles apply to all antigen–antibody reactions & form the basis of many clinical diagnostic tests  OVERVIEW- CLINICAL LABORATORIES PROVIDE A WIDE RANGE OF TEST PROCEDURES THAT ARE THE FOUNDATION OF MODERN MEDICINE I. Epitope Detection by Antibodies III. Epitope Detection in and on Cells 1) Particulate antigens 1) Immunofluorescence (IF) i. Direct agglutination  Monoclonal antibodies ii. Indirect or passive agglutination 2) Flow cytometry 2) Soluble antigens IV. Assessment of Immune Function i. Radial Immunodiffusion 1) Phagocyte function ii. Double-diffusion 2) Proliferation iii. Immunoelectrophoresis (IEP) 3) Cytotoxic T-lymphocyte assay II. Epitope Quantitation by Antibodies V. Assessment of Hypersensitivity 1) Radioimmunoassay 1) Allergy skin testing (type I hypersensitivity) i. Direct RIA ii. Indirect RIA 2) Fluorescent immunosorbent assay (FIA) 3) Enzyme-linked immunosorbent assay (ELISA) EPITOPE DETECTION BY ANTIBODIES  Many clinical lab tests are based on the specificity of antibodies for antigen and their ability to recognize epitopes  Antibody-based assays are epitope-detecting tools and most are based on the quantitative precipitin curve  Examples: A. Particulate antigens 1. Direct agglutination 2. Indirect or passive agglutination B. Soluble antigens 1. Radial Immunodiffusion 2. Double-diffusion 3. Immunoelectrophoresis (IEP)  A. PARTICULATE ANTIGENS  Particulate antigens (e.g. erythrocytes, bacteria, antigen-coated latex beads) are normally evenly dispersed in suspension  Cross-linking of antigen-bearing particles by antibodies disrupts the homogeneity of the suspension leading to clumping of particles (Agglutination)  Examples: i. Direct agglutination- IgM antibodies efficiently cross-link the particles ii. Indirect or passive agglutination- anti-immunoglobulin is used to cross-link antigen-bound antibodies *If particulate antigen is an erythrocyte it is called hemagluttination  I. DIRECT AGGLUTINATION  Usually involves IgM antibodies that cross-link epitopes on cells or particles  IgM- largest immunoglobulin & has 10 epitope-binding site, very efficient at cross-linking epitopes on adjacent particles  Other isotypes are less efficient in direct agglutination (because of smaller size & less binding sites)  Same rules that govern the quantitative precipitin reaction apply to agglutination reactions  Too much antibody inhibits agglutination (equivalent to the zone of antibody excess)  Inhibition of agglutination by antibody is known as the prozone  To prevent prozone effect, dilutions of antibody are added to identical concentrations of particulate antigen  Typically, twofold or serial dilutions of antibody are prepared  Titer- The lowest concentration of antibody that causes agglutination  Relative measures of antibody activity & often expressed as the reciprocal of the dilution (e.g., 1:16, 1:32, 1:64)  II. INDIRECT OR PASSIVE AGGLUTINATION  Used to detect non-IgM antibodies or antibodies in concentrations too low to be detected by direct agglutination  Human antibodies may not directly agglutinate antigen-bearing particles (e.g., bacteria, erythrocytes, latex particles) or show agglutination of very low titer  Sensitivity may be enhanced by the addition of an anti- immunoglobulin reagent (e.g., rabbit antihuman immunoglobulin)  Second-step antibodies- increase binding over a greater span & increase valence by their ability to bind primary antibody B. SOLUBLE ANTIGENS  Epitopes present on soluble molecules will precipitate from solution on reaction with the “right” amount of antibody  Quantitative precipitin reaction requires preparation of several antigen–antibody samples & is too difficult & time-consuming to find application in clinical laboratory  Several Examples:simple modifications allow visualization of immune precipitates in agar (semisolid growth medium) i. Radial immunodiffusion- https://www.youtube.com/watch?v=Bn-w6P_9TUA ii. Double-diffusion iii. Immunoelectrophoresis (IEP)  1. RADIAL IMMUNODIFFUSION (MANCINI TECHNIQUE)  Based on the diffusion of soluble antigen within an agar gel that contains a uniform concentration of antibody  Allows for the rapid and precise determination of the quantity of antigen loaded into the well (quantitative) 1. Antibody-containing molten agar is poured onto glass slide or plastic dish 2. When agar cools & solidifies, wells are cut into the gel matrix, & soluble antigen is placed into the well 3. Antigen diffuses radially from well, forming a precipitin ring at equivalence  Diameter of ring is directly proportional to amount of antigen loaded  Concentration of antigen in a test sample can be accurately determined by comparing its diameter with a standard calibration curve   Specificity of antibody molecules makes them ideal for detection of a wide variety of epitopes  Antibodies or the antigens they detect may be labeled with radioactive molecules, fluorescent molecules, enzymes, or heavy metals  Antibody or antigen binding is then readily detectable & EPITOPE quantifiable  Examples: QUANTITATION 1) Radioimmunoassay- https://www.youtube.com/watch?v=Cs1LvCyEGKg BY ANTIBODIES i. ii. Direct RIA Indirect RIA 2) Fluorescent immunosorbent assay (FIA)- https://www.youtube.com/watch?v=Cs1LvCyEGKg 3) Enzyme-linked immunosorbent assay (ELISA)- https://www.youtube.com/watch?v=c-LyVc_GEhk  1) RADIOIMMUNOASSAY (RIA)  Sensitive but risky- Potential exposure to radioactivity & radioactive waste 1. Direct RIA  Uses radiolabeled antibody or its ligand (antigen)  Antibody is incubated with ligand & unbound reactants are removed from the system (known as phase separation) 2. Indirect RIA  Uses radiolabeled secondary antibody (anti-immunoglobulin) to detect the binding of a primary antibody  A phase separation method must be employed to remove unbound radiolabeled secondary antibody (like direct RIA)  2) FLUORESCENT IMMUNOSORBENT ASSAY (FIA)  Relies on antibodies or their ligands labeled with various fluorescent dyes (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE))  Specific & relatively sensitive  Does not have the hazards associated with radionuclides & RIA  Phase separation of antibody & ligand (antigen) by immobilization of one reactant onto polystyrene prior to addition of fluorochrome- labeled reactant  Bound fluorochrome-labeled reactant is retained if it binds to immobilized reactant & removed by washing if unbound  Fluorescence = binding  3) ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) & ENZYME IMMUNOASSAY (EIA)  Specific & quantitative  Replaced RIA in several tests because it offers advantage of safety & speed  No radioactive decay, so the reagents used are relatively stable  Sensitivity is ≥ RIA or fluorescent immunosorbent assay because an enzyme-labeled reactant is used to turn chromogenic substrate from colorless→color  Color change of the substrate indicates that an enzyme-labeled reactant has bound  Increasing substrate incubation time allows low-concentration enzyme to convert more substrate to enhance test sensitivity (within limits) ELISA VIRTUAL LAB  https://www.biointeractive.org/classroom-resources/immunology-virtual-lab EPITOPE  Epitopes expressed both in & on the surface of cells may be detected by using radiolabeled, enzyme-labeled, DETECTION or fluorochrome-labeled antibodies  Examples: IN AND ON 1. Immunofluorescence Monoclonal antibodies CELLS 2. Flow cytometry 1. IMMUNOFLUORESCENCE (IF)  Uses fluorescent dyes (e.g., FITC) covalently coupled to antibody 1. Thin section of tissue prepared & mounted on a glass slide 2. Section is bathed in a solution containing FITC-labeled antibody (direct) or a solution containing a primary antibody (indirect) & is then washed 1. If indirect, FITC-labeled anti- immunoglobulin is added  Presence of epitopes is visualized with a fluorescent microscope  NEW- REVIEW OF LAST CLASS  Describe the premise of Flow cytometry:  Fluorescent antibodies added to sample of mixed cells  Stream of fluid drops containing antibody labeled cells passed through a laser and detector to see which cells fluoresce  This is then plotted on a two-dimensional dot plot  For visuals: https://www.youtube.com/watch?v=EQXPJ7eeesQ FLOW CYTOMETRY GATING STRATEGY EXAMPLE ASSESSMENT OF IMMUNE  Examples: 1) Phagocyte function FUNCTION 2) Proliferation 3) Cytotoxic T-lymphocyte assay  1. PHAGOCYTE FUNCTION  Functional capacity of phagocytic cells can be assessed by their ability in ingest antibody- or opsonin-coated particles  Phagocyte function can be assessed by incubating phagocytic cells with coated particles (e.g., latex beads or antibody-bound cells) or with bacteria for 30 to 120 minutes  Particle inclusion within the cell is assessed by microscopy  Enzymatic activity of phagocytes can be assessed by measuring the levels of individual degradative or oxidative enzymes (e.g., NADPH oxidase) produced by these cells  2. PROLIFERATION ASSAY  Stimulating lymphocytes to proliferate in response to specific antigen or a substance is used to assess immune function 1. Peripheral blood mononuclear cells (lymphocytes, monocytes, & dendritic cells) are isolated & placed in tissue culture for 48-72 hours 2. A specific stimulator (antigen) that individual may have been previously exposed is added to culture  Alternatively, a nonspecific stimulant (mitogen) is added to assess the ability of a particular subpopulation of leukocytes to respond 3. Radionuclide (such as 3H-thymidine) is added for final 18 to 24 hours of culture 4. Incorporation of 3H-thymidine into DNA is a measure of proliferative ability  3. CYTOTOXIC T-LYMPHOCYTE (CTL) ASSAY  Assessment of ability of cells to induce lysis by # of radiolabeled target cells that are killed 1. Radioactive sodium chromate (Na 251CrO4) readily crosses cell membrane in live cells & binds to cytoplasmic proteins 2. Radiolabeled target cells are washed to remove unbound sodium chromate & target cells are incubated with CD8 + T cells 3. Within 4 hours, CD8+ T cells lyse 51Cr-labeled cells bearing appropriate pMHC class I & release 51Cr-protein complexes into culture medium 4. Intact cells & cellular debris are removed by centrifugation & radioactivity in cell-free medium is used to quantify cytotoxic activity  Can also be used to measure NK or NKT activity ASSESSMENT OF HYPERSENSITIVITY  Hypersensitivity- Immune-mediated damage to host tissues  Four types: 1. Type I (immediate hypersensitivity) 2. Type II 3. Type III 4. Type IV  1. ALLERGY SKIN TESTING (TYPE 1 HYPERSENSITIVITY)  Sensitivities to allergens (antigens) (e.g., pet dander, mold, & pollens [“hay fever”], or certain foods) are common allergic disorders  Arises from development of allergen-specific IgE antibodies on surface of tissue mast cells  Skin testing is a common, convenient, & relatively painless procedure to test an individual’s reaction to an allergen  Usually, a prick/puncture test can be used 1. Diluted allergen is administered by scratching the skin surface (percutaneous) rather than being injected into the dermis 2. Sensitive (atopic) individuals develop wheal-and-flare (redness & swelling) reaction within 20–30 min.  If skin-prick test = negative → intradermal test  A small amount of diluted antigen is injected within the skin  More sensitive than the skin-prick test  Rely on inflammation caused by allergen-IgE induced degranulation of mast cells in the dermis *Risk of severe allergic reaction- have antihistamine or epinephrine available* DR. D’S ALLERGY TEST DR. D’S ALLERGY TEST INTRADERMAL THIS IS WHAT WE CALL A HOME RUN….