Antigen – Antibody Reaction

Antigen–Antibody Reaction Antigen-antibody reaction • Bimolecular association where the antigen and antibody combine with each other specifically and in an observable manner . o Similar to an enzyme-substrate interaction. o Only difference is, it does not lead to an irreversible alteration in either antibody or in antigen. PROPERTIES OF ANTIGEN ANTIBODY REACTIONS 1. Specific: o Ag-Ab reaction involves specific interaction of epitope of an antigen with the corresponding paratope of its homologous antibody. o Exception is the cross reactions which may occur due to sharing of epitopes among different antigens. o In such case, antibody against one antigen can cross react with a similar epitope of a different antigen. PROPERTIES OF ANTIGEN ANTIBODY REACTIONS 2. Non-covalent interactions: o Union of antigen and antibody requires formation of large number of non-covalent interactions between them such as: Hydrogen bonds Electrostatic interactions Hydrophobic interactions van der Waals forces PROPERTIES OF ANTIGEN ANTIBODY REACTIONS • 3. Strength- The strength or the firmness of the association is influenced by the affinity and avidity of the antigen-antibody interaction. o Affinity  Refers to sum total of non covalent interactions between a single epitope of an antigen with its corresponding paratope present on antibody.  Affinity can be measured by two methods- i) by equilibrium dialysis and ii) by surface plasmon resonance metho PROPERTIES OF ANTIGEN ANTIBODY REACTIONS • Avidity: Term used to describe the affinities of all the binding sites when multivalent antibody reacts with a complex antigen carrying multiple epitopes. o When a complex antigen carrying multiple epitopes reacts with a multivalent antibody; the total strength (i.e. avidity) would be much higher than the individual affinity at each binding site. o Avidity is not exactly equal but lower than the sum of all affinities. o This difference is primarily due to geometry of antigen antibody binding. PROPERTIES OF ANTIGEN ANTIBODY REACTIONS o Avidity is a better indicator of strength of an antigen antibody reaction. o Avidity of an antibody can compensate for its low affinity. For example, IgM has a low affinity than IgG but it is multivalent (10 valencies), therefore has a much higher avidity.It can bind to an antigen more effectively than IgG. Diagnostic Use of Ag-Ab reactions • Specific and observable • The diagnostic tests based on antigen-antibody reactions are called as immunoassays • Immunoassays can be broadly categorized into two typeso Antigen detection assays o Antibody detection assays Qualitative assays • Result is read as ‘positive’ or ‘negative’ based on presence or absence of antigen or antibody in the clinical specimen. Disadvantages • Number of antigen or antibody molecules in the reaction are disproportionate to each other. o If either antigen or antibody are present in higher quantity - false negative. To rule out a false negative result, it is ideal to test the series of diluted sera (quantitative test) instead of just testing the one specimen of undiluted serum. Quantitative assays • When the qualitative test turns positive, the exact amount of antibody in serum can be known by serial dilution of the patient’s serum and mixing each dilution of the serum with a known quantity of antigen. • The measurement of antibody is expressed in terms of units or more commonly titer. Quantitative assays • Quantitative tests are more reliable as they can differentiate between true negative and false negative results. Marrack’s or Lattice hypothesis • Ag-Ab reaction is weak or fails to occur when the number of antigen and antibodies are not proportionate to each other. • Prozone phenomenon - In the earlier test tubes, antibodies are excess, hence the Ag-Ab reaction does not occur. • Post zone phenomenon - In the later test tubes, antigen is excess, hence the Ag-Ab reaction fails to occur. Marrack’s or Lattice hypothesis • In prozone/post zone - Lattice does not enlarge, due to inhibition of lattice formation by the excess antibody or antigen respectively, as the valencies of the antibody and the antigen respectively, are fully satisfied. Evaluation of Immunoassays • Sensitivity and specificity are the two most important statistical parameters. • Sensitivity is defined as ability of a test to identify correctly all those who have the disease i.e. true positives. • Sensitivity is calculated as = True positives True positives + False negatives Evaluation of Immunoassays • Specificity is defined as ability of a test to identify correctly all those who do not have disease i.e. true negatives. • Specificity is calculated as = True negatives True negatives + False positives Types of antigen-antibody reactions Conventional techniques - Newer techniques - • Precipitation reaction • Enzyme linked immunosorbent assay (ELISA) • Agglutination reaction • Enzyme linked fluorescent assay (ELISA) • Complement fixation test • Immunofluorescence Assay (IFA) • Neutralization test • Radioimmunoassay (RIA) • Chemiluminescence-linked immunoassay (CLIA) • Immunohistochemistry • Rapid tests Lateral flow assay (Immunochromatographic test)  Flow through assay • Western blot • Immunoassays using electron microscope PRECIPITATION REACTION • Definition-When a soluble antigen reacts with its antibody in the presence of optimal temperature, pH and electrolytes (NaCl), it leads to formation of the antigen-antibody complex in the form of: o Insoluble precipitate band when gel containing medium is used or o Insoluble floccules when liquid medium is used (precipitate remains suspended as floccules) Precipitation in liquid medium • Ring test: In a narrow tube (e.g. capillary tube), antigen solution is layered over an antiserum; o Precipitate ring appears at the junction of two liquids. o Example: Streptococcal grouping by Lancefield technique, and Ascoli’s thermoprecipitin test done for anthrax. Precipitation in liquid medium • Flocculation test- When a drop of antigen is mixed with a drop of patient's serum, then floccules appear. o Examples of slide flocculation test- VDRL and RPR tests used for diagnosis of syphilis o Examples of tube flocculation test- Kahn test used previously for syphilis Precipitation in gel (Immunodiffusion) • Using 1% soft agarose gel for precipitation reaction has many advantages over liquid mediumo Results in formation of clearly visible bands instead of floccules that can be preserved for longer time. o Can differentiate individual antigens from a mixture as each antigen forms a separate band after reacting with specific antibody. Principle of Immunodiffusion • Whether only Ag diffuses (single diffusion) or both Ag and Ab diffuse (double diffusion) • Whether Ag or Ab diffuses in one dimension (i.e. vertical diffusion when test is done on a tube layered with gel) or two dimensions (i.e. diffusion in both X and Y axis when test is done on a slide or a petri dish layered with gel) Types of immunodiffusions in gel 1. Single diffusion in one dimension (Oudin procedure) 2. Double diffusions in one dimension (Oakley- Fulthorpe procedure) 3. Single diffusion in two dimensions (Radial immunodiffusion) 4. Double diffusions in two dimensions (Ouchterlony procedure) Single diffusion in one dimension (Oudin procedure) Double diffusions in one dimension (Oakley- Fulthorpe procedure) Single diffusion in two dimensions (Radial immunodiffusion) Double diffusions in two dimensions (Ouchterlony procedure) Double diffusions in two dimensions (Ouchterlony procedure) • Examples of double diffusions in two dimensions include: o Elek's test for detecting toxin of Corynebacterium diphtheria. o Eiken test to detect toxin of Escherichia coli Precipitation in gel in presence of electric current 1. Electroimmunodiffusion (EID) 2. CIEP (Countercurrent immunoelectrophoresis) 3. Rocket electrophoresis Electroimmunodiffusion • Electric current is applied to a slide layered with gel. • Helps in identification and approximate quantitation of various proteins present in the serum. Counter-current immunoelectrophoresis • Test is even faster (takes 30 minutes) and more sensitive than EID. • Popular in the past - detecting alpha fetoprotein in serum and capsular antigens of Cryptococcus and meningococcus in the cerebrospinal fluid. Rocket electrophoresis • One-dimensional single electroimmunodiffusion test. • Was mainly used in the past for quantitative estimation of antigens. AGGLUTINATION REACTION • Definition-When a particulate or insoluble antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. • Advantage: o More sensitive than precipitation test. o Clumps are better visualized and interpreted than bands or floccules. AGGLUTINATION REACTION • Wide diagnostic applications of agglutination reactions. • Classified as: o Direct agglutination reactions. o Indirect (passive) agglutination reactions. o Reverse passive agglutination reactions. • Performed either on a slide, or in tube or in card or some time in microtiter plates. Direct agglutination test • Antigen directly agglutinates with the antibody. • Slide agglutination: o Performed to confirm the identification and serotyping of bacterial colonies grown in culture. o Method used for blood grouping and cross matching. Tube agglutination • Standard quantitative test for estimating antibody in serum. • Antibody titer can be estimated as the highest dilution of the serum which produces a visible agglutination. Tube agglutination- Applications • • • • Typhoid fever (Widal test) Acute brucellosis (Standard agglutination test) Coombs Antiglobulin test (see below) Heterophile agglutination tests: o Typhus fever (Weil Felix reaction) o Infectious mononucleosis (Paul Bunnell test) o Mycoplasma pneumonia (Cold agglutination test) Microscopic agglutination test (MAT) • Agglutination test is performed on a microtiter plate. • Result is read under a microscope. • MAT is done for leptospirosis. Indirect or passive agglutination test (for antibody detection) • Indirect hemagglutination test (IHA) • Latex agglutination test (LAT) Indirect hemagglutination test (IHA) • Passive agglutination test where RBCs are used as carrier molecules. • IHA was used widely in the past, but is less popular at present. Latex agglutination test (LAT) for antibody detection • Polystyrene latex particles (0.8 – 1 µm in diameter) are used as carrier molecules which are capable of adsorbing several types of antigens. • For better interpretation of result, the test is performed on a black color card. • Drop of patient’s serum (containing antibody) is added to a drop of latex solution coated with the antigen and the card is rocked. Latex agglutination test (LAT) for antibody detection • Positive result is indicated by formation of visible clumps. • LAT is one of the most widely used tests at present as it is very simple and rapid. • Used for detection of ASO (antistreptolysin O antibody). Reverse passive agglutination test (for antigen detection) • Antibody is coated on a carrier molecule which detects antigen in the patient’s serum. Test Carrier molecule Clinical applications RPHA (Reverse passive hemagglutination assay) RBCs • Detection of hepatitis B surface antigen (HBsAg) – not used now. Latex agglutination test Latex particles • Detection of CRP (C reactive protein), RA (rheumatoid arthritis factor), capsular antigen detection in CSF (for pneumococcus, meningococcus and Cryptococcus) and streptococcal grouping. Essentials Reverse passive agglutination test (for antigen detection) Test Coagglutination test Carrier molecule Staphylococcus aureus Clinical applications • Used in past for antigen detection test in the past (e.g. Salmonella antigen detection from blood and urine). • Obsolete at present. Hemagglutination test • Refers to the agglutination tests that use red blood cells (RBCs) as source of antigen. • Various types of hemagglutination tests include: o Direct hemagglutination test o Indirect hemagglutination test Direct hemagglutination test • Serum antibodies directly agglutinate with surface antigens of RBCs to produce a matt. Examples includeo Paul Bunnell test o Cold agglutination test o Blood grouping o Coombs test or Antiglobulin test Coombs test or Antiglobulin test • • • Performed to diagnose Rh incompatibility by detecting Rh antibody from mother’s and baby’s serum. Rh incompatibility is a condition when an Rh negative mother (Rh Ag -ve and Rh Ab -ve) delivers a Rh positive baby (Rh Ag +ve and Rh Ab -ve). During birth, some Rh Ag +ve RBCs may pass from fetus to the maternal circulation and may induce Rh Ab formation in mother and affect future Rh positive pregnancies. Coombs test or Antiglobulin test • • • • Rh antibodies are incomplete or blocking antibodies of IgG type. They can cross placenta and bind to Rh Ag on fetal RBCs. Does not result in agglutination; instead they block the sites on fetal RBCs. Such reaction can be visualized by adding Coombs reagent (antiglobulin or antibody to human IgG) which can bind to Fc portion of Rh Ab bound on RBCs, resulting in visible agglutination. Coombs test or Antiglobulin test - Types • Direct Coombs test • Indirect Coombs test Viral hemagglutination test • In strict sense, it is not an antigen antibody reaction. The hemagglutinin antigens (HA) present on surface of some viruses • Hemagglutinating viruses (e.g. influenza virus) can agglutinate with the receptors present on the surface of RBCs. Technical issues in agglutination reactions • Two main problems pertaining to agglutination - can cause false negative agglutination test. o Prozone phenomenon o Blocking antibodies