Immunochemical Techniques Flashcards
Competitive formats use ____ reagents (Ab or other Ag binder)
Limited
Noncompetitive formats use ____ reagents (Ab or other Ag binder) and are two-site or “____” assays
Excess; sandwich
What does it mean that competitive immunoassays are simultaneous?
All reactants are mixed together simultaneously or at the same time
Simultaneous competitive immunoassay
- Principle
Labeled and unlabeled Ag compete for a limited number of binding sites on an Ab titer
- Avidity for Ab is the same for both
Simultaneous competitive immunoassay
- Proportionality observed b/w bound labeled and unlabeled Ag concentrations
The probability of labeled Ag binding to the Ab binder is inversely proportional to unlabeled Ag
Simultaneous competitive immunoassay
- Examples of tests
- RIA
- FIA
- EIA
Noncompetitive immunoassay
- Principle
A captured Ab is bound to a solid phase. Unlabeled Ag reacts with the solid phase Ab, then there’s a washing step. Labeled Ab is added; it reacts with the unbound Ag on a 2nd and distinct epitope. Another washing step occurs, and the amount of the bound label is determined
Noncompetitive immunoassay
- Proportionality observed b/w bound labeled and unlabeled Ag concentrations
Directly proportional
What is the difference of a heterogeneous immunoassays from homogeneous immunoassays?
- Heterogeneous require a separation step to separate the bound label from the free, unbound reactants
- Homogeneous do not require a separation step
EMIT
- What does it stand for?
Enzyme-Multiplied Immunoassay Technique
EMIT
- What does it detect?
Change in absorbance after adding substrate
EMIT
- Homogeneous or heterogeneous?
Homogeneous
EMIT
- Proportionality
Direct
ELISA
- What does it stand for?
Enzyme-Linked Immunosorbent Assay
ELISA
- What does it detect?
Change in absorbance
ELISA
- Homogeneous or heterogeneous?
Heterogeneous
ELISA
- Proportionality
Direct or indirect
MEIA
- What does it stand for?
Microparticle Enzyme Immunoassay
MEIA
- What does it detect?
Fluorescence
- *MEIA**
- Homogeneous or heterogeneous?
Heterogeneous
MEIA
- Proportionality
Direct
FIA
- What does it stand for?
Fluorescent immunoassays
FIA
- What does it detect?
Fluorescence
FIA
- Homogeneous or heterogeneous?
Heterogeneous
FIA
- Proportionality
Direct or indirect
FPIA
- What does it stand for?
Fluorescence Polarization Immunoassay
FPIA
- What does it detect?
Plane polarized light
FPIA
- Homogeneous or heterogeneous?
Homogeneous
FPIA
- Proportionality
Indirect
FPIA
- Most common fluorescent label
FITC
Chemiluminescence
- What does it detect?
Visible light produced from labels after a base is added and is measured by a luminometer
Chemiluminescence
- Proportionality
Direct
Chemiluminescence
- Two common labels
- Luminol
- Acridinium ester
