Immunoassays Flashcards
Avidity
Overall strength of antibody to antigen binding
Epitope
Specific antibody binding sites found on antigen
Immunoassay
Analytical method that uses antibodies or antigens to measure specific chemicals
What are antibodies?
Immunoglobulins (proteins)
produced by B lymphocytes
Describe the structure of an Immunoglobulin
Two heavy, two light chains covalently bonded (disulfide)
Form a Y shape
Bottom defines the antibody
Top binds to the epitopes
What are the five classes of Antibodies?
IgG: Crosses placenta
IgA: Body secretions (15% of serum IGs)
IgM: First responder
IgE: Low concentration in human serum
IgD: Unknown
What’s another term for Epitope?
Antigenic determinant
What are factors that can affect Avidity?
Intermolecular Forces
Van Der Waals Forces
Hydrophobic Interactions
What’s the difference between Non-Competitive and Competitive Immunoassays?
Non-Competitive binds to epitopes; what is binded is measured
Competitive competes for spots with patient antigen
Type I Immunoassay
Antibody is added with patient antigen
If antigen has correct epitopes, binding will occur
Bound antibody is measured; Antibody is proportional to antigen
Type II Immunoassay
Same as type I but a second antibody is added for confirmation
Antibody is proportional to antigen
Type III Immunoassay
Competitive and Homogenous
patient and reagent antigen compete for spots
When antigen binds, it is deactivated
Free reagent antigen is measured
Type IV Immunoassay
Competitive and Heterogenous
Same as Type III
Binding does not deactivate antigen
Bound and unbound reagent antigen must be separated
Enzyme-Labeled Immunoassay
Enzyme is used to detect Immunoassay reaction
measured by spectrometry
What are the two most common Enzyme -Labeled Immunoassays?
Enzyme-Linked (ELISA)
Enzyme-Multiplied (EMIT)
Enzyme-Linked Immunosorbent Assay (ELISA)
Antibody and patient antigen are added
Second enzyme antibody is added
Antigen that contains both antibodies is sandwiched between
Enzyme activity is proportional to antigen
Enzyme-Multiplied Immunoassay Technique (EMIT)
Competitive
Binding deactivates enzyme antigen
Enzyme activity is proportional to antigen
Fluorescent-Labeled Immunoassay
Competitive
Light excites fluorophore
polarization of emitted light is measured
Chemiluminescent-Labeled Immunoassay
Produced by Luminol and Acridinium
Not deactivated by binding
Particle Immunoassay
Binding of particle-labeled antigen and antibody increases turbidity
Measured by Photometry
Turbidimetry/Nephelometry
