ISLAB-AGABRXN

• ANTIGEN-ANTIBODY REACTION

  • interactions between antigens and antibodies
  • highly specific
  • antigen reacts only with antibodies produced by itself or with closely related antigens
  • antibodies recognize molecular shapes on antigens = antigen (epitopes) and antibody (paratopes)
  • Generally, the better the fit of the epitope in terms of geometry and chemical character to the antibody combining site, the more favorable the interactions that will be formed between the antibody and antigen and the higher the affinity of the antibody for antigen
• SALIENT FEATURES OF AG-AB REACTION

  Specificity of Ag-Ab reaction

  • each antibody binds to specific antigen
  • an interaction similar to lock-and-key.
• SALIENT FEATURES OF AG-AB REACTION

  Immune Complex

  • Ag + Ab = Ag-Ab Complex (basic unit product)
  • molecule formed from the binding of multiple antigens to antibodies
  • the bound antigen and antibody act as a unitary object, effectively an antigen of its own with a specific epitope.
• SALIENT FEATURES OF AG-AB REACTION

  Binding Site of Ag-Ab reaction

  • the antibody attaches with the antigen
  • the part of the antigen which combines with antibody is called epitope (antigenic determinant), part of the antigen that is recognized by the immune system, specially by antibodies (B or T cells)
  • the part of the antibody that recognizes the epitope is called a paratope.
• BINDING FORCE OF AG-AB REACTION

  Closeness between antigen and antibody

  • the better the fit, the greater the strength of bind
  • when they are apart, binding strength is low.

  Non-covalent bonds or intermolecular forces

  • the bonds that hold the antigen to the antibody combining site are all non-covalent in nature.
  • hydrogen bonds, electrostatic force, Van der Waals forces, hydrophobic bonds
• BINDING FORCE OF AG-AB REACTION

  Affinity of antibody

  • Affinity = measure of binding strength at single binding site; the strength of the reaction between a single antigenic determinant and a single combining site on the antibody.
  • Avidity = measure of total binding strength of a unit or complex.
• APPLICATION OF AG-AB REACTION

  • determination of blood groups for transfusion
  • Serological exposure to infectious agents
  • development of immunoassays for the quantification of various substances
  • to detect the presence or absence of protein in serum
  • determining the characteristics of certain immunodeficiency disease
• TYPES OF AG-AB REACTIONS

  • agglutination
  • precipitation
  • complement fixation
  • ELISA
  • immunofluorescence
  • radioimmunoassay
  • immunofixation
  • immuno-electrophoresis
• TYPES OF AG-AB REACTIONS

  Agglutination

  • when a particular antigen is mixed with its antibodies in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated.
  • the antibody of the serum causes the cellular antigen to form clumps and these are called agglutinins.
  • the particulate antigens that are aggregated are termed agglutinogens.
• TYPES OF AG-AB REACTIONS = AGGLUTINATION

  Slide Agglutination

  • rapid method to determine the presence of agglutinating antibodies.

  Procedure

  • to a uniform suspension of particulate antigen, a drop of saline is added and a drop of antiserum is added.
  • The 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. Sometimes confirmation may be done by observing slide under microscope.
  • This is the test used for blood grouping and cross-matching.
• TYPES OF AG-AB REACTIONS = AGGLUTINATION

  Tube Agglutination

  • the standard method for quantitative estimation of antibody.
  • The serum containing antibody is diluted serially with saline in several small test tubes, to which a constant volume of antigen 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.
• TYPES OF AG-AB REACTIONS = AGGLUTINATION

  Principle

  • Particulate antigen antibody
  • Lattice formation (clumping) = antigen binds with Fab sites of two antibodies forming bridges between antigens.
• TYPES OF AG-AB REACTIONS = AGGLUTINATION

  • Direct agglutination = blood bank
  • Passive hemagglutination = antigen reagent produced by treating RBC with tannic acid to allow adsorption of protein antigens.
  • Passive latex agglutination = antigen in reagent is attached to latex particle.
• TYPES OF AG-AB REACTIONS = AGGLUTINATION

  Agglutination Inhibition

  • Step 1 = patient serum (antigen) is reacted with limited amount of antibody reagent.
  • Step 2 = indicator is added (same antigen for which you are testing bound to RBC or latex carrier particle)
  • Positive test (no agglutination) = If patient has antigen for which you are testing, the reagent antibody will be bound in step and unavailable to react with the indicator
  • Negative test (agglutination) = If patient does not have the antigo, reaprint antibody is not bound in step and is available to react with indicator.
• TYPES OF AG-AB REACTIONS = AGGLUTINATION

  Examples of inhibition reactions

  • hemagglutination inhibition test for rubella
  • Latex agglutination inhibition test for other viruses.
• TYPES OF AG-AB REACTIONS

  Precipitation

  • When a soluble antigen combines with its antibodies in the presence of an electrolyte (NaCl) at a particular temperature and pH it forms an insoluble precipitate of Ag-Ab complex.
  • the antibody causing precipitation is called precipitin.
• TYPES OF AG-AB REACTIONS = PRECIPITATION

  Principle

  • Soluble antigen + antibody (proper proportions)
  • Lattice formation (visible precipitate) = antigen binds with Fab sites of two antibodies.

  Examples

  • Double diffusion (ouchteriony)
  • Single diffusion (radial immunodiffusion)
  • Immuno-electrophoresis
  • Immunofixation
• TYPES OF AG-AB REACTIONS

  Complement Fixation

  • lysis of RBC or bacteria requires non-specific unstable components of fresh serum which are called complement.

  Limitations

  • serum must be heat inactivated
  • Stored serum becomes anti-complementary
  • elaborate QC and standardization required
  • only used for IgM antibodies
• TYPES OF AG-AB REACTIONS = COMPLEMENT FIXATION

  • Step 1 = antibody and entire allowed to combine in presence of complement.
  • Step 2 = indicator is added (suspended RBC coated with hemolysin)
  • Positive test (no hemolysis) = if complement is bind in step 1, it will not be available to combine with indicator, no hemolysis occurs.
  • Negative test (hemolysis) = complement was not bound in step 1 and is available to react with indicator, hemolysis occurs.
• TYPES OF AG-AB REACTIONS

  Enzyme-linked Immunoassay (ELISA)

  • sandwich technique
  • Monoclonal or polyclonal antibody adsorbed on solid surface (head or microtiter well)
  • Add patient serum: If antigen is present in serum, it binds to antibody-coated bead or well
  • Add excess enzyme-labeled antibody (antibody conjugate): forms antigen-antibody-labeled antibody "sandwich" (antibody in conjugate is directed against another epitope of antigen being assayed)
  • Add enzyme substrate, incubate and read absorbance
• TYPES OF AG-AB REACTIONS

  Enzyme-linked Immunoassay (ELISA)

  • Washing required between each step. Improper washing leads to false positive results.
  • Absorbance is directly proportional to antigen concentration.

  Examples:

  • HIV testing
  • Serum HCG (pregnancy)
  • Tests for hepatitis antigens and antibodies
  • antibodies to bacteria and viruses
• TYPES OF AG-AB REACTIONS

  Immunofluoresence

  • Fluorescence = the property of absorbing light rays of one particular wavelength and emitting rays with a different wave length.
  • Fluorescent dyes = show up brightly under UV light as they convert into visible light.
• TYPES OF AG-AB REACTIONS

  Immunofluoresence

  • Direct = add fluorescein-labeled antibody to patient tissue, wash, and examine under fluorescent microscope.
  • Indirect = add patient serum to reagent (tissue containing known antigen), wash, add fluorescein-labeled antiglobulin, wash, and examine under fluorescent microscope.

  Examples

  • Testing for Anti-nuclear Antibodies (ANA)
  • Fluorescent Treponemal Antibody Test (FTA-AL)
• TYPES OF AG-AB REACTIONS

  Radioimmunoassay (RIA)

  • very sensitive and specific
  • can be used for detecting antigen or antibody (detects antigen, but using known antigen to detect serum antibody is possible)
• TYPES OF AG-AB REACTIONS = Radioimmunoassay (RIA)

  Competitive Binding Assay

  • Patient antigen and labeled antigen are incubated with known amount of specific antibody (unlabeled and labeled anti compete for binding with antibody, Radioly REA) to detect.
  • Wash to remove unbound antigen quantitate the set of antigen (analy) is complete.
  • Radioactivity counted on a gamma counter, compare to standard curve.
  • Results

  The lower the radioactive count, the higher the concentration of unlabeled antigen (patient).
• LATTICE

  • A series of points that are arranged in a distinct pattern.

  Nonlattice (More Sensitive) = immunoassays

  • radial immunoassay (RIA)
  • enzyme immunoassay (EIA)
  • fluorescent immunoassay (FIA)
  • nephelometry
• LATTICE = less sensitive

  • Counter Current Immunoelectrophoresis
  • Complement Fixation
  • Agglutination
  • Flocculation (precipitation)
  • Rocket Electrophoresis
  • Radial immunodiffusion (RID)
  • Ouchteriony (double immunodiffusion)
  • immunofixation (IFE)
  • Immunoelectrophoresis (IEP)