Presentation on theme: "2. Basic Immunologic Procedures"— Presentation transcript:
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 antigenSensitization cannot be visualizedMultitude of laboratory methods have been developed to make this reaction visible
3 Factors Which Affect Reactions Concentrations of reactantsTemperatureLength of incubationpH of test system
4 Three Distinct Phases of Antigen/Antibody Reactions Primary Phenomenon – SensitizationSecondary Phenomenon – Lattice formationTertiary Phenomenon – Detected by affect on tissues or cells.
5 Primary phenomenonSensitization – binding of antibody to antigen – not visible
6 Primary phenomenonSensitization – binding of single antibody to single antigen siteThese tests areDifficultComplexExpensiveRequire special equipmentTime consuming
8 Secondary PhenomenonSensitization taken a step further to lattice formationFab of Antibody molecule binds to two separate antigens on adjacent antigensIf antigen on large structures such as RBCs causes agglutination.If both antibody and antigen are soluble results in precipitation
14 Secondary Phenomena Most Frequently Utilized Precipitation – soluble antibody reacts with soluble antigenAgglutination – particulate antigens bound together by antibody to form visible complexComplement Fixation – antibody binding to antigen triggers activation of complement, results in cell lysis
15 Antigen-Antibody Binding Union of antigen and antibody requiresAffinityAvidityAffinity 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.
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.
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 ActionGoverns 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 negativePostzone – antigen excess, antibody coats antigen but cannot get lattice formation, results in false negativeZone of Equivalence – antigen and antibody present in optimal proportions to bind and give visible reaction