Transcript antigens
بسم هللا الرحمن الرحيم Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201) Over View Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: [email protected] Clinical Immunology & Serology Practice (Code: MLIS 201) Teaching Objectives: 1- To know the elements of serological reactions 2- To define serological reactions 3- To describe the basics of primary and secondary serological reactions ANTIGENS Definition: Any molecular structure that when introduced is capable of Antibody production. Parts of Antigen Carrier portion - responsible for the molecular weight of antigen Epitope or Determinant - determines specificity of antigen Two Properties of Antigens 1. IMMUNOGENICITY - inherent ability of a substance to induce the specific immune response resulting in the formation of immune lymphocytes or antibodies Factors influencing Immunogenicity (Summary) 1-Foreigness : Foreign substances are immunogenic 2- Molecular size: High molecular weight increase immunogenicity 3- Chemical structure complexity: High complexity increase immunogenicity 4- Route of administration: Parenteral routes are more immunogenic to oral route SC>IP>IV>Intragastric Factors influencing Immunogenicity (Summary) (cont.) 5- Degradability of the immunogen 6-Genotype of the recipient 7- immunogen dose: Appropriate dose Low dose High dose optimum antigenicity low- zone tolerance high-zone tolerance 8- Adjuvant: Substance when injected with an immunogen enhance immunogenicity 2. ANTIGENICITY/SPECIFICITY - the ability to react specifically with the antibody or cell that caused it to be produced ANTIBODIES Definition: Spec. glycoproteins, produced by plasma cells in response to antigenic stimulation of B lymphocytes. Immunoglobulins. Structure of Immunoglobulin 1.Four (4) polypeptide chains: 2 identical LIGHT chains and 2 identical HEAVY chains. 2.Both light and heavy chains are held together by DISULFIDE BONDS. 3.Heavy chains are interconnected by DISULFIDE bonds in the HINGE region. 4.Ig has 2 terminal regions: Carboxyterminal - with constant amino acid sequence (constant region). Aminoterminal - with varying antibody specificity (variable region) Classes of Immunoglobulins IgG IgA IgM IgD IgE Molecular Weight Daltons 150 T 160-400 T 900 T 180 T 190 T Halflife 21-23 days 5-6 days 5 days 2-3 days 2-3 days Subclasses 4 2 2 - - Domains 4 4 5 4 5 Activate Complement? Yes Alt pathway only Yes No. No. IgG Other Name Serum Ig Other can cross Notes placenta except -major Ig in 2º Immune response IgA Secretory Ig predominant Ig in secretion -with J chain -exist as monomerin serum and dimer in secretion IgM Pentameric Ig IgD ___ IgE Reagenic Ig Predominat involved asso. in 1ºimmune in B cell With response activation allergy - binds -has J chain basophils and mast cells elevated during parasitic infections Complement System COMPLEMENT SYSTEM - A set of serum proteins that play a role in cytolytic destruction of cellular antigen by specific antibody. - reaction is nonspecific to the target cell - destroyed at 56ºC for 30 minutes - the in vitro study of antigen-antibody reaction - laboratory study of the activities of the components of blood serum that contributes to immunity Immunologic Reactions: Primary - combination of Ag-Ab; non-visible reaction Secondary - demonstrable Ag-Ab rxn (e.g. precipitation, agglutination) PRIMARY IMMUNOLOGIC TESTS. IMMUNOASSAYS Ligand - any substance that will complex to another substance; (the substance to be measured. A. FLUORESCENCE IMMUNOASSAY Fluorescent Probes used: a. FITC (Fluorescein isothiocyanate) b. Phycocyanin c. Texas red d.Tetramethyl rhodamine -emits green light***mostly used -emits red light -emits red light -emits red orange light Techniques: 1. Direct/Single Layer Immunofluorescent Assay 2. Indirect/Double Layer Immunofluorescent Assay positive FA test for rabies (Image: Centers for Disease Control) negative FA test for rabies (Image: Centers for Disease Control) C. ENZYME IMMUNOASSAY colorimetric reaction Enzymes Used: a. b. c. d. ALP Horseradish Peroxidase Glucose oxidase B-galactosidase Techniques: 1. Direct 2. Indirect 3. Sandwich/Double Ab 4. Competitive Binding 5. Enzyme Inhibition ELISA: DOUBLE ANTIBODY TECHNIQUE Secondary tests PRECIPITATION AGGLUTINATION COMPLEMENT FIXATION NEUTRALIZATION I.PRECIPITATION RXNS - Antigens involved are soluble antigens. Types of Precipitation reaction: 1. Single diffusion, Single Dimension Px serum w/ soluble Ag (+) rxn - formation of pptn line Gel/Agar impregnated w/ known Ab 2. (RADIAL IMMUNODIFFUSION) - uses a plate containing agar with known antibody - Px serum is placed on the wells - Diameter of the pptn line is directly proportional to the concentration of the target antigen 5. Immunoelectrophoresis (IEP) - useful procedure for the ID of monoclonal proteins (Bence Jones Protein) - utilizes both double diffusion and electrophoresis a. Ags migrate under an electric current (-) Ag (+) b. Rgt. Ab Added. Diffusion through gel (-) (+) Antibody c. (+) rxn - Pptn arcs formed (-) (+) STEP 1. STEP 2. II. AGGLUTINATION REACTIONS - Ags involved are particulate antigens. Types of Agglutination Rxn 1.Direct Agglutination (e.g. Blood Typing) 2. Antiglobulin Technique (CoombsTest)/Indirect Agglutination - anti-human IgG is added to bridge the gap between the cells - to demonstrate incomplete antibodies 3.Passive Agglutination - A soluble Ag is artificially attached to a particulate carrier (e.g. cells, latex, bentonite, celloidin, or charcoal 6. Hemagglutination - Agglutination of rbc due to antibody, viruses, bacteria, or other biologic substance. - It is not the antigen of RBC but the artificially attached Ag (after undergoing tx) that are made to react with the Ab - ex. TPHA Study Questions: 40 Compare between: Different Classes of Immunoglobulins Assignment Write notes on A- Primary Ag-Ab reactions شروق ابو الحسن – شروق كمال – غادة عز الدين – فاطمة على – مروة اشرف B- Secondary Ag-Ab reactions صالح ميلود – محمد زغلول – محمد فوزى – محمود محمد رمضان Thanks