W7.4 Flashcards
Types of Immunoassays
”- Enzyme immunoassays
- Chemiluminescence assays
- Immunofluorescence assays
“
Heterogeneous Labeled Immunoassays
”- involve solid phase (microwell, bead) and requires washing steps to remove bound antigen or antibodies
- Competitive (competition between original antigen and “add-in” antigen) or noncompetitive “
Homogeneous Labeled Immunoassays
”- consists of only a liquid phase and does not require washing steps
- Competitive only
“
Enzyme Immunoassay (EIA)
“Also known as, enzyme-linked immunosorbent assay or ELISA
- Designed to detect either antigen or antibody by producing an enzyme-triggered color change (colorimetric)
- Utilizes microtiter plates
“
EIA functions/steps
”- Uses the catalytic properties of enzymes to detect and quantitate immunologic reactions
- Enzyme reacts with the substrate; creating a color change
- Most common detection enzymes include: horseradish peroxidase (HRP), alkaline phosphatase (ALP)(Note that these same enzymes are used in other types of labeled immunoassays)
- Enzyme must have:
- High degree of stability
- Extreme specificity”
Types of EIA
Direct, Indirect, Sandwich, Competitive
Direct EIA
may be used directly on specimen
Indirect EIA
uses a labeled secondary antibody for detection
Sandwich EIA
designed to detect a specific type of antibody
Competitive EIA
“used to measure concentration of antigen (or antibody)
“
Sandwich : Direct and Indirect EIA
”- “Sandwiches” the antigen between 2 antibodies (a capture antibody and a detection antibody)
- Antigen must have 2 antigenic epitopes that can bind antibody “
Sandwich : Direct and Indirect EIA (Basic steps)
”- Plate is coated with a capture antibody; and blocked
- Sample is added; antigen binds
- Detecting antibody is added; binds antigen
- Enzyme-linked secondary antibody is added; binds detecting antibody
- Or may have the direct method and does not use this secondary antibody
- substrate is added “
EIA advantages
”- Good sensitivity
- Does not require expensive equipment: read by spectrophotometry
or noted the presence or absence of color
- Inexpensive reagents with a long shelf-life
“
EIA disadvatages
”- Specimens may have inhibitors
- Enzyme reactions are sensitive to temperature
- Cross-reactivity resulting in nonspecific signal
EIA results
yields 3 different type of data : Quantitiative, Qualitative, Semiqualitative
