Immunological Techniques PDF

11. Immunological techniques B2 tags Immunology Recall questions Review 📌 The antibody immobilised: capture ab The secondary ab: detection ab 1...

11. Immunological techniques B2 tags Immunology Recall questions Review 📌 The antibody immobilised: capture ab The secondary ab: detection ab 1. Basics of Antigen-Antibody Reactions Nature of Ag/Ab Reactions Lock and key concept Non-covalent bonds involved in Ag/Ab reactions: Hydrogen bonds Electrostatic bonds Van der Waals forces Hydrophobic bonds Characteristics of Ag/Ab reactions: Multiple bonds: These bonds collectively provide strong binding despite being individually weak. Reversible nature Binding Mechanisms High-affinity vs. low-affinity interactions Affinity: 11. Immunological techniques 1  Strength of a single antigenic determinant (epitope) binding to an antibody site Avidity: The overall strength of the binding between an antibody and an antigen, taking into account multiple binding sites ~Velcro™ effect, where multiple weak interactions combine to create a strong overall attachment. Avidity is more relevant to biological systems than affinity Specificity vs. Cross-Reactivity Specificity: The ability of an antibody to exclusively recognize a single antigenic determinant (epitope). Specific antibodies are critical for precise immune responses. Cross-reactivity: Occurs when an antibody reacts with similar or shared epitopes on different antigens. This can lead to immune responses against unintended targets. 2. Factors Affecting Ag/Ab Reaction Measurements Affinity and avidity Ag:Ab ratio (antigen/antibody) Physical form of the antigen: soluble antigens vs particulate antigens Soluble antigens: small, dissolve in solution. When bind with abs, it can remain in solution or precipitate if the concentration is high enough (ex: a fragment of virus, viral protein…) Particulate antigens: antigens that are part of/is a larger surface (ex: blood, viruses…), often have mutliple epitopes; when bind to ab, they form visible aggregates, a process called agglutination. Equivalence (lattice formation during binding): At the optimal antigen- antibody ratio, a lattice forms, leading to visible precipitation. 3. Types of Antibodies Monoclonal antibodies Polyclonal antibodies Definition homogenous population of abs heterogenous population of abs which are produced by a single which are produced by a single 11. Immunological techniques 2  Monoclonal antibodies Polyclonal antibodies clone of plasma B cells clone of plasma B cells particular epitope on a particular different epitopes on a particular Interact with antigen antigen In vivo: The animal is immunized In vitro: fusing a specific B cell with an antigen, ⇒ activation of with a myeloma cell (long-lived B- various B cells, each producing Production cell tumor) to create a hybridoma. antibodies against different This hybridoma cell line produces epitopes of the antigen ⇒ collect identical antibodies (monoclonal). antiserium Likelihood of cross reactivity for Reactivity Low chance of cross reactivity different antigens sharing similar/same epitopes widely used for therapies, can be limited use: suitable for pathogen modified for targeted functions detection (Retains binding sites while Applications altering other parts) ⇒ Covalent Not reproducible: Each bleed attachment of drugs, dyes, (antiserum taken) can be enzymes, beads, toxins… different Tests Based on Antigen-Antibody Reactions Can detect either antigen or antibody: Using antigens to detect antibodies Using antibodies to detect antigens Types: Qualitative tests: Presence or absence of agglutination Quantitative tests: Measurement of titers or concentrations Agglutination and Hemagglutination Tests 11. Immunological techniques 3  📌 Hemagglutination: clumping of red blood cells Agglutination: The clumping of particles, such as cells or beads, caused by the binding of specific antibodies to antigens on the surface of these particles. Principle: Endpoint is the visible clumping of particulate antigens 1. Passive agglutination: Soluble antigens coated onto particles (ex: beads) + serum ⇒ detect antibodies in the serum 2. Coombs (Antiglobulin) tests: Direct: Antiglobulin added to RBC. If agglutination occurs, it indicates the presence of antibodies or complement proteins attached to the RBCs. Indirect: The patient's serum is mixed with red blood cells that have known antigens on their surface. Incubate to allow binding. Antiglobulin is added. Agglutination indicates the presence of antibodies in the serum. Applications: Detection of anti-Rh ab Diagnose autoimmune hemolytic anemia 3. Agglutination inhibition tests: inhibit agglutination due to competition with a soluble antigen (Ag) to detect presence of viruses and antiviral antibodies in patient. Virus and RBCs: A known amount of virus is mixed with a suspension of red blood cells. 11. Immunological techniques 4  Sample Addition: The patient's serum (which may contain antibodies) is added to the mixture. Incubation: The mixture is incubated to allow any antibodies in the serum to bind to the virus. Observation: If antibodies are present, they will inhibit the virus from causing hemagglutination, resulting in no clumping of red blood cells. If antibodies are absent, the virus will agglutinate the RBCs, causing visible clumping. Applications Blood typing Bacterial infections: detect the presence of specific antibodies in a patient's serum that bind to bacterial antigens, causing visible clumping Viral infections (e.g., hemagglutination inhibition for influenza) 11. Immunological techniques 5  Precipitation Tests Principle: Formation of visible precipitates in solution or gel matrices Equivalence Zone: The formation of a visible precipitate occurs when the antigen and antibody are in optimal proportions. This is known as the equivalence zone. If there is excess antigen or antibody, precipitation may not occur. Methods: 1. Radial Immunodiffusion (RID): (quantitative) Procedure: Antigens are placed in a well cut in an agarose gel containing antibodies. As the antigen diffuses outwards, it forms a precipitate ring with the antibodies. The diameter of the ring is proportional to the concentration Application: Used to measure the concentration of specific antigens in a sample. 2. Immunoelectrophoresis (qualitative analysis of antigen mixtures) Procedure: Combines electrophoresis and immunodiffusion. Antigens are separated by electrophoresis, and then antibodies are added to form precipitate arcs. Application: Used to analyze complex mixtures of antigens and to detect multiple specific antibodies. 3. Countercurrent electrophoresis (qualitative) Ag and Ab migrate toward each other by electrophoresis Used only when Ag and Ab have opposite charges When they meet in optimal proportion, precipitate line is formed ⇒ Rapid detection of antigens and antibodies Applications: 11. Immunological techniques 6  After precipitation, we can use the precipitate for: Purification of soluble proteins (by SDS gel electrophoresis) Analyzing antibody or antigen presence (by western blotting) Radioimmunoassays (RIA) Competitive Unlabeled antigens from the sample compete with radiolabeled antigens for binding to specific, known number of antibodies ⇒ The number of free radiolabeled antigens is inversely proportional to the number of antigens from patient Non-competitive Ag detection Immobilize Ab Incubate with sample Add radioactively labeled antibody Amount of labeled Ab bound is proportional to the amount of Ag in the sample Applications: Sensitive detection of proteins, hormones, and antigens Limitations Replaced by enzyme-linked methods in most applications 8. Enzyme-Linked Immunosorbent Assay (ELISA) Principle: Uses enzyme-conjugated secondary antibodies ⇒ add substrate and measure color change to detect antigens or antibodies Types Indirect ELISA: Detects antibodies; uses antigen-coated plates 11. Immunological techniques 7  Sandwich ELISA: Detects antigens; uses antibodies-coated plates Competitive ELISA: Measures antigen concentration; involves competition between sample antigen and labeled antigen. Variations: Biotin-conjugated antibodies can be used with streptavidin- conjugated enzymes to amplify signals ELISPOT: measure the number of cytokine-secreting cells in a population. Applications: Detection and quantification in research and diagnostics 9. Western Blot (Immunoblotting) Combines electrophoresis and immunoassays to detect specific proteins Used for: Verifying immunoprecipitation results Quantifying and identifying proteins 10. Immunochromatography Principle: Separation and detection of antigens/antibodies using lateral flow techniques Method Sample Application: When the sample is applied, it moves through the conjugate pad, where any target antigens in the sample bind to the colored particle-coated antibodies. Migration: The complex (antigen bound to the colored particle-coated antibodies) migrates along the test strip. Test Line Binding: At the test line, the immobilized antibodies capture the antigen-bound complexes. This accumulation of colored particles forms a visible line, indicating a positive result. 11. Immunological techniques 8  Control Line: Ensures the test is working properly by capturing excess colored particles, forming a visible line whether or not the test is positive. Examples: Rapid diagnostic tests (e.g., pregnancy tests, COVID-19 antigen tests) 11. Immunological techniques 9