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2. Basic Immunologic Procedures

2. Basic Immunologic Procedures. Terry Kotrla, MS, MT(ASCP)BB. Introduction. 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

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2. Basic Immunologic Procedures

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  1. 2. Basic Immunologic Procedures Terry Kotrla, MS, MT(ASCP)BB

  2. Introduction • 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

  3. Factors Which Affect Reactions • Concentrations of reactants • Temperature • Length of incubation • pH of test system

  4. Three Distinct Phases of Antigen/Antibody Reactions • Primary Phenomenon – Sensitization • Secondary Phenomenon – Lattice formation • Tertiary Phenomenon – Detected by affect on tissues or cells.

  5. Primary phenomenon • Sensitization – binding of antibody to antigen – not visible

  6. Primary phenomenon • Sensitization – binding of single antibody to single antigen site • These tests are • Difficult • Complex • Expensive • Require special equipment • Time consuming

  7. Primary Phenomenon Tests • Techniques include: • Immunofluorescence • Radioimmunoassay • Enzyme immunoassay

  8. 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

  9. Secondary Phenomenon Reactions

  10. 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

  11. Secondary Phenomenon • Examples of tests: • Precipitation • Agglutination • Complement Fixation

  12. Tertiary Phenomenon • Reaction not visible, detected by affect of reaction on tissues or cells. • Tests include: • Inflammation • Phagocytosis • Deposition of immune complexes • Immune adherence • Chemotaxis • http://www.cellsalive.com/mac.htm

  13. Phagocytosis

  14. 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

  15. Antigen-Antibody Binding • Union of antigen and antibody requires • Affinity • Avidity • Affinity and avidity determined by Law of Mass Action

  16. 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.

  17. Affinity

  18. Avidity • 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.

  19. Avidity

  20. 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.

  21. 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.

  22. 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.

  23. 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

  24. Precipitation Curve

  25. Precipitation Curve

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