IMMUNE

• Precipitation Reactions


  Involves combining soluble antigen with soluble antibody to produce insoluble complexes that are visible
• Agglutination Reactions


  Process by which particulate antigens such as cell aggregate to form larger complexes when a specific antibody is present

  
• Combination of antigen with specific antibody plays an important role in the laboratory in diagnosing many different diseases

  
• IMMUNOASSAYS


  Assays developed to detect either antigen or antibody, based on the principles of precipitation or agglutination

  
• Affinity


  Initial force of attraction that exist between single Fab site and a single epitope on the corresponding antigen
• Avidity


  Sum of all attractive forces between an Ag and Ab, dictates the overall stability of the Ag-Ab complex
• Precipitation


  1. Combining soluble antigen with soluble antibody to produce insoluble complexes that are visible

  2. First noted in 1897 by Kraus

  3. Law of Mass Action: All antigen–antibody binding is reversible and free reactants are in equilibrium with bound reactants

  
• Precipitation Curve


  Maximum precipitation occurs when Ag and Ab concentration have an optimum ratio<|>Zone of Equivalence: Ag and Ab are equal, therefore max precipitation occur<|>Prozone: More Ab (patient) excess than Ag, remedy is serum dilution<|>Post zone: Ag excess, less Ab (Patient) may lead to false negative, remedy is to repeat the test after a week to give time for antibody production
• Precipitation Reactions


  • Precipitation in a fluid medium
  • Precipitation by a passive immunodiffusion
  • Precipitation by electrophoretic techniques
• Turbidimetry


  Measure of the turbidity or cloudiness of a solution, measures the reduction in light intensity due to reflection, absorption, or scatter
• Nephelometry


  Measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension, the amount of light scattered is an index of the solution's concentration
• Precipitation by a passive immunodiffusion

  1. Ag-Ab complex may be observed in a support media (Agarose gel)

  2. Passive: No electric current is used to speed up reaction of the Ag and Ab combination, but through DIFFUSION

  3. Factors affecting Rate of Diffusion: Size of the particles, Gel viscosity, Temperature, Amount of hydration
• Radial Immunodiffusion


  1. Ab is uniformly distributed in support gel and Ag is applied to a well cut into gel

  2. Single Diffusion-Single Dimension (Oudin): Ag diffuses through the gel, containing immobilized Ab forming insoluble Ag-Ab Complexes

  3. Single Diffusion- Double Dimensions (Mancini, Fahey and McKelvey): Ag diffuses through the gel and ring ppt expands from the well

  4. Ouchterlony Double Diffusion: Both Ag and Ab diffuse independently through a semisolid medium in 2 dimensions
• Electrophoresis


  Technique in which molecules with a net charge are separated when an electric field is applied
• Rocket Immunoelectrophoresis (Laurell Technique)


  Ag is pushed through Ab containing gel under influence of an applied electric field, when they are at equivalence, precipitation will occur forming a cone/ rocket shape band
• Crossed Immunoelectrophoresis (Ressler's Method)

  Proteins are separated by electrophoresis, then subjected to a 2nd electrophoresis where they will move through an Ab-containing agarose gel until a rocket is formed (Ag-Ab reach equivalence)
• Counter Immunoelectrophoresis (Countercurrent electrophoresis)

  Ag and Ab are added to separate parallel wells cut out in an agar gel, when an electric field is applied, the Ag will migrate to the Anode and Ab to the cathode, zone of equivalence will form a precipitate
• Classic Immunoelectrophoresis (Grabar and Williams)


  Ag is introduced in a well and an electric field is applied resulting in separation of proteins, Ab is introduced in an trough parallel to the separated protein, Ag-Ab complex form

  
• Agglutination


  Process by which particulate antigens such as cell aggregate to form larger complexes when a specific antibody is present

  
• Major categories of agglutination reactions

  • Direct/ Active Agglutination
  • Indirect/Passive
  • Reverse Passive
  • Antiglobulin Test
  • Quantitative Assays
• Direct/ Active Agglutination


  Reaction is due to an Ag-AB reaction where in the Ag is inherent native to the cell<|>Aggregation of indicator red blood cells are NOT due to Ag-Ab reactions
• Viral Hemagglutination


  Virus can stick to agglutinate RBCs, not due to Ag-Ab reactions
• Viral Hemagglutination Inhibition (HAI)


  Patient serum incubated with viral particles, viral particles will bind to the Fab region of Anti-viral Abs, indicator RBCs added, positive result is inhibition or absence of agglutination
• Indirect/ Passive Agglutination


  Reactions where Ag has been affixed or absorbed to a carrier/inert particle
• Reverse Passive Agglutination


  Antibody is bound to the carrier, fluid is detected for the presence of Ag

  
• Latex Particle Agglutination Inhibition


  Patient sample (Ag) incubated with Ab in test kit, complex will form if the patient sample contains the corresponding Ag and the Fab sites are no longer available for the Ag-coated latex particles
• Anti-globulin test


  Direct: Detects IgG Ab bound to Ag on Red cells (in-vivo)

  Indirect: Detects presence of Abs in the serum that is still to be attached to an analyte

  
• Indicator Labeled Immunoassays


  Designed for antigens and antibodies that may be small in size or present in very low concentrations, the presence is determined indirectly by using a labeled reactant to detect specific binding

  
• Fluorescence Serology


  Utilizes fluor or fuorochrome that have the ability to absorb light at shorter wavelengths and emit light waves at a visible spectrum/longer wavelengths, covalently linked to Ig (direct) or to anti-globin (indirect) to detect the presence of Ag or Ab

  
• Direct Immunofluorescent Test


  Ag from pt sample fixed to a microscopic slide, fluorescent labelled Ab added, Ab binds to Ag and becomes visible

  
• Antibodies


  May be small in size or present in very low concentrations
• Determining presence of antigens or antibodies


  Use a labeled reactant to detect whether or not specific binding has taken place

  
• Labels


  • Fluorescent
  • Radionuclide
  • Enzyme
  • Free radical
  • Ferritin
• Fluorescence


  Ability to absorb light at shorter wavelengths and emit light waves at a visible spectrum/longer wavelengths
• Fluorescent labels


  Covalently linked to Ig (direct) or to anti-globin (indirect) to detect the presence of Ag or Ab
• Fluorescent labels


  • FITC (Fluorescein isothiocyanate, green color)
  • Lissamine rhodamine B (Tetramethyl rhodamine (TRITC) with fluorescence of color red)
  • DANSYL
  • Algal proteins
• Direct Immunofluorescent Test


  1. Ag from pt sample fixed to a microscopic slide

  2. Fluorescent labelled Ab added

  3. Ab binds to Ag and becomes fixed to slide

  4. Washing to remove unattached Ab

  5. Positive test: Fluorescence
• Indirect Immunofluorescent Test/ Indirect Fluorescent Antibody (IFA)


  1. Ag spread on slide and Pt serum (Ab) added and incubated and washed

  2. Fluorescent antihuman globulin added incubated and washed

  3. If the Pt serum contained the corresponding Ab it would be fixed to the Ag on the slide

  4. Tagged Rgt reacts with the Ab fixed to the Ag

  
• Inhibition Immunofluorescence Test


  1. The Ag is fixed to the slide and then flooded with pt serum

  2. If corresponding Ab is present in the serum, it will attach) fix to the Ag

  3. When specific fluorescent Ab reagent is added it cannot become fixed because all of the antigenic sites are already occupied by the unlabeled pt Ab

  4. Labelled rgt washed off

  5. Positive test: No fluorescence

  
• Complement Staining IF Test

  1. Patient serum and complement mixed with Rgt Ag fixed on the slide

  2. Incubate and Wash

  3. Fluorescent Labelled Anti-Complement is added

  4. Positive: Fluorescence