2. Basic Immunologic Procedures Terry Kotrla, ms, mt(ascp)BB

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2. Basic Immunologic Procedures

  • Terry Kotrla, MS, MT(ASCP)BB


  • Detection of antigen/antibody reactions difficult

  • Can measure EITHER antigen or antibody.

  • Sensitization is the binding of a specific antibody to its’ specific antigen

  • Sensitization cannot be visualized

  • Multitude of laboratory methods have been developed to make this reaction visible

Factors Which Affect Reactions

  • Concentrations of reactants

  • Temperature

  • Length of incubation

  • pH of test system

Three Distinct Phases of Antigen/Antibody Reactions

  • Primary Phenomenon – Sensitization

  • Secondary Phenomenon – Lattice formation

  • Tertiary Phenomenon – Detected by affect on tissues or cells.

Primary phenomenon

  • Sensitization – binding of antibody to antigen – not visible

Primary phenomenon

  • Sensitization – binding of single antibody to single antigen site

  • These tests are

    • Difficult
    • Complex
    • Expensive
    • Require special equipment
    • Time consuming

Primary Phenomenon Tests

  • Techniques include:

    • Immunofluorescence
    • Radioimmunoassay
    • Enzyme immunoassay

Secondary Phenomenon

  • Sensitization taken a step further to lattice formation

  • Fab of Antibody molecule binds to two separate antigens on adjacent antigens

    • If antigen on large structures such as RBCs causes agglutination.
    • If both antibody and antigen are soluble results in precipitation

Secondary Phenomenon Reactions

Secondary Phenomenon

  • These tests are:

    • Easy to perform
    • Less expensive
    • Less time consuming
    • Do not require special equipment
  • Downside is

    • Less sensitive
    • Less specific
    • More interference

Secondary Phenomenon

  • Examples of tests:

    • Precipitation
    • Agglutination
    • Complement Fixation

Tertiary Phenomenon

  • Reaction not visible, detected by affect of reaction on tissues or cells.

  • Tests include:

  • http://www.cellsalive.com/mac.htm


Secondary Phenomena Most Frequently Utilized

  • Precipitation – soluble antibody reacts with soluble antigen

  • Agglutination – particulate antigens bound together by antibody to form visible complex

  • Complement Fixation – antibody binding to antigen triggers activation of complement, results in cell lysis

Antigen-Antibody Binding

Antibody Affinity

  • Describes the strength of a single Ag-Ab bond.

  • As Ag and Ab come close together a chemical bond forms which is weak and can dissociate.

  • How well the Ab fits the Ag will determine stability of bond, “lock and key” fit has strongest affinity.

  • Ab may react with structurally similar Ags, results in cross reactivity.

  • Most antibodies have a high affinity for their antigens.



  • Describes the combined strength of multiple Ag-Ab bonds.

  • Initially bond is easily broken, but multiple bindings at the same time the dissociation is overcome by the sheer number of bonds remaining.

  • Avidity is influenced by both the valence of the antibody and the valence of the antigen.


Affinity versus Affinity

  • Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site whereas avidity refers to the overall strength of binding between multivalent antigens and antibodies.

IgM and IgG

  • Most frequently detected immunoglobulins.

  • IgM has low affinity but high avidity due to 10 binding sites.

  • IgG has 2 strong binding sites, high affinity and avidity.

Law of Mass Action

  • Governs the reversibility of the antigen-antibody reaction.

  • Reversible reaction, visible reaction occurs when the rate of binding exceeds the rate of dissociation.

  • As affinity and avidity increases, strengthens reaction.

Precipitation Curve

  • Depends on concentration of Ag and Ab.

  • Prozone – antibody excess, many antibodies coat all antigen sites- results in false negative

  • Postzone – antigen excess, antibody coats antigen but cannot get lattice formation, results in false negative

  • Zone of Equivalence – antigen and antibody present in optimal proportions to bind and give visible reaction

Precipitation Curve

Precipitation Curve

Kataloq: mlt -> ser
ser -> -

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