Antigen-Antibody Reactions PDF: Types, Applications & Reactions

ANTIGEN – ANTIBODY N REACTIONSR A C E L LA V I CHARLES ANRE VILLACERAN, RMT Antigen–Antibody reactions The interactions between antigens and antibodies Reactions are highly specific, and an antigen reacts only with antibodies produced by it...

ANTIGEN – ANTIBODY N REACTIONSR A C E L LA V I CHARLES ANRE VILLACERAN, RMT Antigen–Antibody reactions The interactions between antigens and antibodies Reactions are highly specific, and an antigen reacts only with antibodies produced by itself or with closely related antigens. Antibodies recognize molecular shapes (epitopes) on antigens. A N Generally, the better the fit of the epitope (in terms of geometry and chemical character) to the E R antibody combining site, the more favorable the interactions that will be formed between the antibody C and antigen and the higher the affinity of the antibody for antigen. A IL L V SALIENT FEATURES OF AG-AB REACTION Specificity of antigen – antibody reaction - each antibody binds to a specific antigen; an interaction similar to lock and key Immune complex - Ag + Ab 🡪 Ag-Ab complex. N - molecule formed from the binding of multiple antigens to antibodies. The bound antigen and A antibody act as a unitary object, effectively an antigen of its own with a specific epitope. Binding site of Antigen – Antibody reaction E R C - In Ag-Ab reaction, the Ab attaches with the antigen. A IL L - The part of the antigen which combines with antibody is called Epitope (also known as antigenic determinant), part of the antigen that is recognized by the immune system, specifically by Antibodies, B cells or V T cells. - The part of the antibody that recognizes the epitope is called a paratope SALIENT FEATURES OF AG-AB REACTION Binding Force of Antigen – Antibody reaction (3 factors) Closeness between ag and antibody - when antigen and antibody are closely fit, the strength of binding is great. When they are apart binding strength is low A N Non-covalent bonds or intermolecular forces - the bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These E R include hydrogen bonds, electrostatic bonds, Van der Waals forces and Hydrophobic bonds AC IL L Affinity of antibody – antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. V Properties of Antigen – Antibody Reaction 1. Antibody affinity Affinity and avidity are both measures of binding strength. 2. Antibody avidity affinity - measure of the binding strength at a single binding site, avidity - is a measure of the total binding strength 3. Cross reaction Application of Ag-Ab reaction Determination of blood groups for transfusion Serological exposure to infectious agents A N Development of immunoassays for the quantification of various substances E R C To detect the presence or absence of protein in serum A IL L Determining the characteristics of certain immunodeficiency disease. V  TYPES OF Ag-Ab REACTIONS 1. AGGLUTINATION 2. PRECIPITATION A N 3. COMPLEMENT FIXATION E R 4. ELISA/EIA AC 5. I IMMUNOFLUORESCENCE L L 6. V RADIOIMMUNOASSAY TYPES OF Ag-Ab REACTIONS Agglutination – When a particular Ag is mixed with its Ab’s in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. The Ab of the serum causes the cellular Ag’s to form clumps and these are called Agglutinins. The particulate antigens that are aggregated are termed Agglutinogens. N Slide agglutination – rapid method to determine the presence of agglutinating antibodies A Procedure E R C To a uniform suspension of particulate Ag, a drop of saline is added and then a drop of antiserum is added. The LA slide is gently rocked or a fine loop is used to mix the contents. If granulation occurs the test is positive. It takes a minute for the test to complete and is visible to the naked eye. Some times confirmation may be L V I done by observing slide under microscope. This is the test used for blood grouping and crossmatching Tube agglutination This is a standard method for quantitative estimation of Ab. The serum containing Ab is diluted serially with saline in several small test tubes, to which a constant volume of Ag suspension is added. A control tube is kept which has no antiserum. The tubes are incubated until visible agglutination is observed. The tube showing highest agglutination is referred to as the titer. Tube agglutination is employed for the serological diagnosis of typhoid, brucellosis and typhus fever. TUBE AGGLUTINATION SLIDE AGGLUTINATION  A N E R AC IL L V  A N E R AC IL L V TYPES OF Ag-Ab REACTIONS Precipitation – when a soluble Ag combines with its Ab in the presence of an electrolyte (NaCl) at a particular temperature and pH, it forms an insoluble precipitate of Ag-Ab complex. The Ab causing precipitation is called Precipitin and the reaction is called precipitation reaction A N E R AC IL L V  TYPES OF Ag-Ab REACTIONS COMPLEMENT FIXATION - refers to the process where complement proteins, part of the innate immune system, bind to and become activated when antibodies and antigens interact. specific antibodies. A N - This process is crucial for immune defense against pathogens and is used in laboratory assays for detecting I. E R The complement system consists of a series of proteins (C1 to C9) that, when activated, lead to the destruction of pathogens, inflammation, and immune cell recruitment. Complement fixation involves the activation of this cascade. AC II. assays. IL L The complement system can be activated through the classical pathway, where antigen-antibody complexes trigger the activation of complement proteins. This is the primary pathway used in complement fixation III. V Complement fixation assays are used to detect antibodies against specific antigens. If the patient’s serum contains the target antibody, the complement system will bind and activate, "fixing" the complement to the antigen-antibody complex. IV. In a diagnostic setting, if a patient has the antibody of interest, the complement is "fixed" to the antigen-antibody complex. If there is no antibody present, complement remains free in the solution. The absence of complement fixation can be detected by a secondary reaction, such as the lysis of red blood cells. COMPLEMENT FIXATION Clinical Applications: >Complement fixation tests are used to diagnose infections (e.g., syphilis, fungal infections, and viral diseases) by detecting specific antibodies in patient serum. immune response. A N >The degree of complement fixation can help determine the titer of antibodies, which indicates the strength of the E R Outcome Interpretation: AC Positive result: Indicates the presence of the antibody in the sample, as the complement has been activated and fixed. IL L Negative result: If there is no antibody present, complement remains unactivated and is free in the solution, indicating a negative result. Advantages and Limitations V The complement fixation test is highly sensitive and specific but may require technical skill and proper controls. False positives and negatives can occur, so it is often used in conjunction with other diagnostic methods.  A N E R AC IL L V TYPES OF Ag-Ab REACTIONS Enzyme Linked Immuno Assay - Diagnostic test that uses an enzyme linked to an antibody or antigen to A N detect the presence and concentration of specific molecules. - Relies on the enzymatic conversion of a substrate to produce a measurable signal, often a color change. E R AC PRINCIPLE IL L V The assay works by using an enzyme-conjugated antibody or antigen to bind to the target molecule. When the substrate is added, the enzyme catalyzes a reaction that produces a detectable signal (color change, fluorescence, or luminescence), which correlates with the amount of target molecule present. KEY COMPONENTS OF EIA 1. Antigen: The substance that is being detected (e.g., virus, bacteria, protein). A N 2. Antibody: The molecule that binds specifically to the antigen, often linked to an enzyme. E R AC 3. Enzyme: The enzyme (e.g., horseradish peroxidase or alkaline IL L phosphatase) catalyzes the reaction that generates the detectable signal. V 4. Substrate: The chemical that reacts with the enzyme to produce a measurable signal.  TYPES OF ELISA I. Direct ELISA: The antigen is directly attached to the surface, and the enzyme-labeled antibody binds to it to generate a signal. A N II. Indirect ELISA: The antigen is attached to the surface, but a primary antibody (specific for the antigen) binds to it, followed by a secondary antibody linked to the enzyme to amplify the signal. E R III. Sandwich ELISA: The antigen is "sandwiched" between two antibodies: one AC coated on the surface and the other labeled with the enzyme. This is IL L particularly useful for detecting large molecules like cytokines or hormones. > “SANDWICH” because the target antigen is "sandwiched" between two V antibodies. The first antibody (capture antibody) is attached to the plate, and the second antibody (detection antibody) binds to the antigen, forming a layered structure, just like a sandwich. IV. Competitive ELISA: The antigen and a labeled antigen compete for binding sites on the antibody. The more antigens in the sample, the less signal is generated. INTERPRETATION OF RESULTS N Positive Result: A color change or fluorescence indicates the presence A of the target molecule. E R C Negative Result: No color change or weak fluorescence indicates the A IL L absence or low concentration of the target molecule. V Quantification: Results are often reported in terms of concentration by comparing sample values to a standard curve.  ADVANTAGES AND LIMITATIONS ADVANTAGES i. ELISA is highly sensitive and specific, allowing for the detection of small quantities of analytes. ii. A N Provides quantitative results, making it suitable for determining concentration. iii. R Can analyze multiple samples simultaneously, making it ideal for large-scale testing. E iv. C Can be used to detect a wide range of substances (antibodies, antigens, hormones, proteins). A LIMITATIONS IL L i. ii. iii. V The assay involves several steps, which may lead to errors if not performed carefully. Cross-reactivity, improper controls, or sample handling can result in inaccurate results. While ELISA is relatively straightforward, it can take time to perform, especially when preparing samples and developing the assay. All ELISAs are EIAs, but not all EIAs are ELISAs. Some EIAs use different formats, such as membrane-based or rapid tests, while ELISA specifically refers to plate-based assays. A N E R C EIA is a broader term that includes all immunoassays that use A IL L enzymes to detect antigen-antibody reactions. V ELISA is a specific type of EIA that is performed on a solid surface (like a microplate) and usually involves washing steps to remove unbound substances. TYPES OF Ag-Ab REACTIONS IMMUNOFLUORESCENCE - technique that uses fluorescent dyes (fluorophores) attached to A N antibodies to visualize the presence and location of specific antigens E R in cells or tissues under a fluorescence microscope. AC PRINCIPLE IL L V Antibodies bind to specific antigens, and a fluorescent dye attached to the antibody emits light under UV or specific wavelengths, making the antigen-antibody interaction visible under a microscope. Types of Immunofluorescence A. Direct Immunofluorescence (DIF) A fluorophore-labeled primary antibody binds directly to the target antigen. >Advantage: Faster and more specific. A N E R >Use: Detecting tissue-bound antibodies in autoimmune diseases (e.g., lupus, pemphigus). AC L L B. Indirect Immunofluorescence (IIF) I V A primary antibody binds to the antigen, and a secondary antibody (labeled with a fluorophore) binds to the primary antibody. >Advantage: More sensitive due to signal amplification. >Use: Detecting antibodies in patient serum (e.g., ANA test for autoimmune disorders). Common Fluorophores Used 1. FITC (Fluorescein Isothiocyanate) → Emits green fluorescence. 2. 3. Rhodamine → Emits red fluorescence. A N DAPI (4′,6-Diamidino-2-Phenylindole)→ Binds DNA, emits blue fluorescence. E R C Interpretation of Results A IL L Positive Result: Fluorescent signal indicates the presence of the target antigen or antibody. V Negative Result: No fluorescence means the target antigen or antibody is absent.  A N E R AC IL L V TYPES OF Ag-Ab REACTIONS RADIOIMMUNOASSAY (RIA) - Technique that uses radioactive isotopes to measure A N antigen-antibody interactions. It is extremely sensitive and can detect E R very low concentrations of analytes in biological samples. AC PRINCIPLE IL L V A radioactive antigen competes with the sample's antigen for antibody binding; the less radioactivity detected, the more antigen is present, which is measured using a standard curve. KEY COMPONENTS OF RIA 1. Antigen: The molecule being measured (e.g., hormone, virus, protein). A N 2. Antibody: Binds specifically to the antigen. E R 3. Radioactive Label (Tracer): A labeled form of the antigen, usually using Iodine-125 (¹²⁵I). AC IL L 4. Separation Method: To remove unbound components, common V methods include precipitation or adsorption techniques. ADVANTAGES AND LIMITATIONS OF RIA ADVANTAGES ✔ High Sensitivity → Detects very low concentrations of analytes. A N ✔ Specificity → Uses antigen-antibody binding, ensuring accurate detection. ✔ Quantitative → Provides precise measurements for clinical and research applications. E R AC LIMITATIONS IL L ❌ Radiation Hazard → Requires strict safety measures due to radioactive materials. V ❌ Short Shelf Life → Radioisotopes decay over time, reducing effectiveness. ❌ Costly and Complex → Requires specialized equipment (gamma counter) and regulatory approval.  A N E R AC IL L V RIA VS ELISA Feature RIA ELISA Label Used Radioactive isotopes (¹²⁵I) A N Enzymes (e.g., HRP, ALP) Detection Gamma counter E R Colorimetric reaction Sensitivity Higher AC High but slightly lower Safety IL L Radioactive risk Safer, no radiation Cost V Expensive and regulated Cheaper and widely used A lattice is a series of points that are arranged in a distinct pattern. A N E R AC IL L V