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Immunology Methods

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Immunology Methods Powered By Docstoc
					          CLS 420 Clinical Immunology &
              Molecular Diagnostics




Immunologic Methods

        Part 2
    Basic Methods
                       Objectives
• Explain the principle of each method presented, and give
  a clinical use for each.
• Contrast precipitation, agglutination and flocculation,
  including:
   –   Reaction time and conditions
   –   Antigen state
   –   Immunoglobulin class
   –   Lattice formation
• Describe heat inactivation of patient serum, including
  method and purpose.
• Discuss general reasons for performing immunologic
  tests.
Precipitation Based Methods

   Soluble antigen combines with
 antibody to form aggregates which
      precipitate out of solution.
               Nephelometry
• Antibody reagent is
  combined with patient
  sample.
• If antigen is present in
  the patient’s sample,
  Ag/Ab complexes will
  form and precipitate
  out of solution.



       Click on image at right wait for animation to begin.
                 Nephelometry
• When light is passed
  through the solution, the
  precipitates cause the
  light to scatter at various      Light
  angles.                          source
• The light that is scattered
  at a particular angle is
  measured. This
  corresponds to the level
  of antigen in the sample.
                                Detector
           Flocculation
Uses fine particles of antigen to detect
    antibody in patient’s serum.




Negative test                     Positive test


  Click on images above wait for animation to begin.
          Double Immunodiffusion
           Ouchterlony Method
• Testing performed in
  agar gel.
• Antigen is placed in
  one well.
• Antibody is place in      AG
  other well.
• Each diffuses
  through gel.
• If antibody is specific        AB
  to that antigen, a
  precipitin line will
  form where the 2
  meet.
Double Immunodiffusion
        Identity




  Click on image above wait for animation to begin.
Double Immunodiffusion
     Nonidentity




 Click on image above wait for animation to begin.
  Double Immunodiffusion
      Partial Identity
D antigen

 Da     Db

             D             Da
             AG            AG
 Dc     Dd



                  Anti-D
Immunofixation Electrophoresis
•   Proteins separated by electrophoresis
•   Antiserum (antibody) is applied to the gel.
•   Ag/Ab complexes form in the gel.
•   The gel is stained to reveal precipitin bands.




                     Application point
       Anode        Patient serum         Cathode

    (+ electrode)                        (- electrode)
             IFE stained gels
      = Serum application point




SPE     Anti-IgG    Anti-IgA      Anti-IgM Anti-Kappa Anti-Lambda
           Western Blot
                                    p24
                                    gp 41




                                    gp120/160


Negative     Patient         Positive
Control      specimen        Control

No bands     Patient bands compared to Pos Control
Agglutination Based Methods

  Antibodies cause the cross-linking
    of particulate antigen, usually
            found on a cell.
            Direct Agglutination
• The antigen is a natural
  part of the solid’s surface.
• Often performed at room
  temperature.
• May use centrifugation to
  bring antigen and
  antibody into closer
  proximity.
• Can be used to detect
  antigen or antibody


            Click on image at right wait for animation to begin.
      Passive Agglutination
Antigens on a carrier molecule, such as latex,
combine with patient’s sample for antibody detection.




         Click on image above wait for animation to begin.
Reverse Passive Agglutination
Antibody is bound to the carrier molecule, which is
then mixed with patient’s sample to detect antigen.




           Click on image above wait for animation to begin.
      Inhibition of Agglutination
• Antibody reagent is combined
  with patient’s specimen.
• If patient’s specimen contains
  the antigen for that antibody,




                                                Y
  they will react.                                                 Y
• Reagent antigen is added.




                                                             Y
• A positive reaction will show                   Y
  no agglutination, because the
  antibodies were bound to the
  patient antigen before the
  reagent antigen was added.
• A negative reaction shows
  agglutination between reagent
  antibodies and antigen.
           Click on images at right wait for animation to begin.
Other Methods
         Neutralization
Positive Test                          Negative Test




 The presence of an antibody prevents the
    antigen from functioning correctly.


  Click on images above wait for animation to begin.
          Complement Fixation
• The patient’s serum is heated at 56oC for 30 minutes to
  inactivate any complement present.
• Patient’s treated serum is incubated with known antigen
  and a known quantity of guinea pig complement.
• If the patient has an antibody to the antigen, they will
  react and the complement will bind to the Fc pieces of
  the antibodies.
• Sheep RBCs that are coated with hemolysin are added.
• The test is incubated, centrifuged and read for
  hemolysis.
• In a positive test, the complement will have been used
  up by the patient’s antibody, and no hemolysis will be
  present.
 Complement Fixation
Negative test                       Positive test




   Click on images above wait for animation to begin.
  “Labeled” Methods
 Attaches a “tag” to either the
antigen or antibody. This “tag”
can be detected and measured.
      Parts of a labeled assay
•   Analyte (labeled and unlabeled)
•   Specific antibody
•   Separation of bound and free
    components
•   Detection of label
•   Standards/calibrators
            Classification
• Heterogeneous: Method that requires a
  step that separates bound analyte from
  unbound analyte.
• Homogeneous: Method that does not
  require a separation step.
                Competitive EIA
• Enzyme labeled
  antigen competes with
  unlabeled patient
  antigen for binding
  sites on fixed
  antibodies.
• A chromogen is added
  that reacts with the
  enzyme.
• The level of color
  development is
  inversely proportional
  to the level of patient
  antigen.

           Click on image at right wait for animation to begin.
       Capture (Sandwich) EIA
• Patient’s sample is
  incubated with bound
  antibody.
• Following a wash, a
  second antibody that is
  labeled with a
  chromogen is added.
• The level of color
  development
  corresponds with the
  amount of antigen
  “captured”.

           Click on image at right wait for animation to begin.
Enzyme-multiplied Immunoassay
      Technique (EMIT)




   Y         Y           Y          Y
•This is a homogeneous competitive binding assay.
•Color development is _______proportional to the
concentration of antigen.
     Direct Fluorescence
Negative test                              Positive test




  Fluorescently labeled antibody is used to detect
              antigen fixed to a slide.

      Click on images above wait for animation to begin.
     Indirect Fluorescence




Positive Test                              Negative Test
•Known antigen fixed to slide
•Patient’s serum added (unknown antibody)
•Incubation & wash
•Fluorescently labeled anti-human globulin reagent added.

        Click on images above wait for animation to begin.
        Microparticle Capture
•   Uses microbeads coated with known
    antigen or antibody.
•   The beads are incubated with a
    fluorescently labeled analyte and the
    patient’s sample.
•   The test mixture is centrifuged (or
    magnetized) to collect the beads, which
    are then analyzed for fluorescence.
        Fluorescent Polarization
• Free labeled antigen excited
  by polarized light emits
  unpolarized light.
• Labeled antigen/antibody         Y
  complexes excited by
  polarized light emit polarized
  light.
• FPIA is a competitive binding
  assay in which labeled antigen
  competes with unlabeled
  (patient) antigen for antibody
  binding sites.
• The more labeled antigen that
  is bound to antibody, the more
  polarized light is emitted.
          Chemiluminescence
•   Uses chemical labels that, when
    oxidized, produce a substance of a
    higher energy level.
•   When this substance decays to its
    original state, it emits energy in the form
    of light.
    – Common label materials include:
      •   Luminol
      •   Acridium esters
      •   Peroxyoxalates
Comparing Antibody
   Quantities
   Antibody titers
             Antibody Titer
• An antibody titration can help determine
  antibody concentration levels.
• Twofold serial dilutions of serum
  containing an antibody are made, then
  tested against cells possessing the target
  antigen.
• The titer is the reciprocal of the greatest
  dilution in which agglutination is observed.
Two-fold Serial Dilutions
Tube       1 2 3               4       5       6       7       8       9       10 11 12


           0     0.2   0.2     0.2     0.2     0.2     0.2     0.2     0.2     0.2     0.2      0.2
Saline           ml    ml      ml      ml      ml      ml      ml      ml      ml      ml       ml



           0.2   0.2   0.2     0.2     0.2     0.2     0.2     0.2     0.2     0.2     0.2      0.2
Serum      ml    ml    ml of   ml of   ml of   ml of   ml of   ml of   ml of   ml of   ml of    ml of
                       tube    tube    tube    tube    tube    tube    tube    tube    tube     6%
                       2       3       4       5       6       7       8       9       10       BSA

RBC        0.1   0.1   0.1     0.1     0.1     0.1     0.1     0.1     0.1     0.1     0.1      0.1
Suspen     ml    ml    ml      ml      ml      ml      ml      ml      ml      ml      ml       ml
sion


Final      1:1   1:2   1:4     1:8     1:16    1:32    1:64    1:128   1:256   1:512   1:1024   control

Dilution
                  Results
• Titers provide more valuable information
  when tested in parallel with a previous titer
  specimen.
• A comparison of the current specimen’s
  results and previous specimen’s current
  results should be made.
• A change in titer of 2 or more tubes is
  considered to be significant.
      Reasons to perform a titer
•   Acute and convalescent
•   Prenatal
•   Verify past infection
•   Confirm vaccination
Primary vs. Secondary Humoral
          Response
                                    IgG




             IgM

                                     IgM

                   IgG


  First                  Second
  exposure
                         exposure
Congratulations
   You have finished
Immunology Student Lab
       Lectures!
Good Luck on your exam!

				
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