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

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

• 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:

• Inflammation
• Phagocytosis
• Deposition of immune complexes
• Immune adherence
• Chemotaxis

• http://www.cellsalive.com/mac.htm

Phagocytosis

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

• Union of antigen and antibody requires

• Affinity
• Avidity

• Affinity and avidity determined by Law of Mass Action

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.

Affinity

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.

Avidity

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