Principles and Concepts of Immunological Methods by Geoffrey Kwenda, BSc (Hons), MSc, PhD - Department of Biomedical Sciences School of Health Sciences University of Zambia-unza

11/16/2020 Geoffrey Kwenda, BSc (Hons), MSc, PhD Department of Biomedical Sciences School of Health Sciences University of Zambia Principles and Concepts of Immunological Methods July 16, 2020 Definition of Immunology • Immunology is the study of our protection from foreign macromolecules or invading organisms and our responses to them. —Latin - Immunis = “exempt” —English = protection from disease Immunology • Recognition of self and non-self —Antigens • Elimination of non-self —Exogenous targets Microbes Allergens Foreign material —Endogenous targets Tumours Two Arms of Host Defence Innate immunity • Natural immunity • Defence system functional at birth • Preformed or available within hours after infection • Pattern recognition • Widely present in nature • Adaptive immunity – Acquired – Available within days – Specificity – Memory – In higher vertebrates Innate Adaptive Key Players in Immunology Innate Adaptive Cells • Phagocytes • Epithelial Cells • NK Cells • Lymphocytes (B-cells, T-cells) Defence Proteins • Complement • Antimicrobial (poly)peptides • Antibodies •Innate Immunity: Fast-acting Less specific recognition Early during evolution e.g. barriers to infection such as skin and mucus surfaces •Adaptive Immunity: Specificity Distinguish antigens sometimes present from those always present Memory and Recall •Cells of the immune system Leukocytes originating from bone marrow stem cells •Communication with other systems Endocrine system Central nervous system Skeletal system •Disruption of the Immune System Hypersensitivity Autoimmunity Immunodeficiency Psychoneuroimmunology Overview of Immune System 11/16/2020 Manifestations of Infection Clinical presentation of infectious disease reflects interaction between host and microorganism Interaction affected by host immune status and microbial virulence factors Signs and symptoms vary according to site of infection and severity of infection (acute or chronic?) Physician may predict course of disease and likely cause by combining epidemiological clues with signs and symptoms Immunological techniques are used to: •Detect, identify and quantify Ag in clinical samples •Evaluate Ab response to infection •Evaluate Person’s history of exposure to infectious agents Specificity of Ab-Ag interaction and sensitivity of many immunological techniques make them powerful tools In most cases same technique can be used to evaluate a person’s Ag and Ab status Immunological techniques Antigen + Antibody Immune Complex Methods of Detection 1. Precipitation of soluble Ag • Immunodiffusion • Immunoelectrophoresis 2. Agglutination of cell-bound Ag 3. Immunolabelling • Radioimmunoassay • Enzyme-linked assays • Immunohistochemistry • Leukocyte assays • Flow Cytometric analysis and Luminex 4. Rosette & plaque formation 5. Immunogenetics 6. Antibody production 7. Evaluation Methods 1.Precipitation of soluble Ag • Precipitation techniques are based on ability of most Ab preps to interact with more than one epitope on a protein or infectious agent fact that each Ab molecule interacts with more that one Ag Ab diffuse and meet Ag At appropriate conc visible ppte forms Stain ppte (eg for protein) 11/16/2020 a) Immunodiffusion A A A B B B Zone of equivalence + Precipitation Contaminant present in antigen Present in reduced concentration Within limited concn range for both Ag and Ab, termed EQUIVALENCE ZONE, Ab cross-links Ag into complex that is too large to stay in soln and therefore pptes Increasing Antigen Concentration Amount of Precipitin Antigen Excess (Postzone) Equivalence Antibody Excess (Prozone) i) Ouchterlony (Qualitative Double Diffusion) • Double diffusion precipitation tests involve the diffusion of Ags and Abs in a soft agar gel, forming zones of precipitation where they meet • Characterise relationship between 2 antigens • If antigens are in the same precipitation line ,they fail to cross Qualitative (Double Diffusion): Ouchterlony Limitations : Insensitive Needs large unit of Ab and Ag Resolve by CIE – placing agar in electric field ii. Quantitative: Radial Immunodiffusion Measurement of IgG concentration in serum by single radial immunodiffusion. The diameter of the standards (0) enables a calibration curve to be drawn and the concentration of IgG in the sera under test can be read off: T1-serum from patient with IgG myeloma; 15mg/ml, T2- serum from patient with hypogammaglobulinaemia; 2.6 mg/ml, T3-normal serum; 9.6 mg/ml Demerits •Only 1 Ag can be tested at a time •Method slow 11/16/2020 b) Immunoelectrophoresis • Migration of serum proteins in gel is combined with precipitation by Ab • Used to study complicated Ag mixtures • Ag separated by electrophoresis i. Qualitative ii. Quantitative: Rocket electrophoresis Ag is electrophoresed into gel containing Ab. Distance …