Labelled immunoassays Flashcards
Purpose of labelled immunoassays.
Detects antigens or antibodies in small sizes or low concentrations.
Key mechanism of competitive labelled immunoassays.
Labeled antigen competes with patient antigen; result inversely proportional to analyte concentration.
Key mechanism of noncompetitive labelled immunoassays.
Capture antibody binds patient antigen; result directly proportional to analyte concentration.
Characteristic of heterogeneous immunoassays.
Requires washing step to separate bound from free analyte.
Characteristic of homogeneous immunoassays.
No separation step required; simpler but less sensitive.
Major disadvantage of heterogeneous immunoassays.
Complexity and high cost.
Discovery of radioimmunoassay (RIA).
Developed by Yalow and Berson in the 1950s.
Primary label used in RIA.
Radioactive isotopes.
Common isotopes in RIA.
Iodine-131, Iodine-125, Hydrogen-3, Carbon-14.
Device for measuring radioactivity in RIA.
Scintillation counter.
Analyte concentration relationship in competitive RIA.
Radioactivity inversely proportional to analyte concentration.
Analyte concentration relationship in noncompetitive RIA.
Radioactivity directly proportional to analyte concentration.
RIA clinical utility.
Highly sensitive for detecting drugs or hormones.
Health risks of RIA.
Radioactive exposure, potential mutagenesis, and carcinogenicity.
Discovery of fluorescent immunoassay.
Albert Coons, 1941.
Key label in fluorescent immunoassay.
Fluorophores or fluorochromes.
Outcome of a positive fluorescent immunoassay.
Fluorescent light emission proportional to antigen/antibody concentration.
Most commonly used fluorophore.
FITC (Fluorescein Isothiocyanate), emits green light.
Fluorophores emitting red light.
Phycocyanin, Texas Red.
Fluorophore emitting red-orange light.
Tetramethyl rhodamine.
End result of a fluorescent immunoassay.
Emission of fluorescent light.
Target and labeled reagent in direct immunofluorescent assay.
Target: Antigen; Labeled reagent: Antibody.
Applications of direct fluorescent immunoassays.
Diagnosis of viral diseases (HSV, CMV, EBV), detection of cell surface antigens, Chlamydial antigens, and in flow cytometry.
Target and labeled reagent in indirect immunofluorescent assay.
Target: Antibody; Labeled reagent: Antihuman-globulin (AHG).
Applications of indirect immunofluorescent assays.
FTA-ABS, FANA testing.
Principle of fluorescence polarization immunoassay (FPIA).
Based on the change in polarization of fluorescent light emitted from a labeled molecule when bound by an antibody.
Relationship between fluorescence polarization and analyte concentration in FPIA.
Inversely proportional to analyte concentration.
Instrument used in fluorescence polarization immunoassay.
Polarization analyzer.
Advantage of fluorescence polarization immunoassay.
More sensitive and specific.
Disadvantage of fluorescence polarization immunoassay.
Quenching decreases the intensity of fluorescence.
Label used in enzyme immunoassay (EIA).
Enzymes.
Common substrates used in enzyme immunoassays (EIA).
HPO, ALP, and Glucose oxidase
Commonly used enzyme in enzyme immunoassays (EIA).
ALP (Alkaline Phosphate), prepared from calf intestine; Horseradish Peroxidase from horseradish
Enzyme used in enzyme immunoassays (EIA) that catalyzes glucose oxidation.
Glucose oxidase.
Enzyme used in enzyme immunoassays (EIA) for breaking down lactose.
B-galactosidase.
Types of breakdown products in enzyme immunoassays (EIA).
Chromogenic, fluorogenic, or luminescent products are produced.
End reaction of enzyme immunoassay (EIA).
Colorimetric reaction with chromogenic substrate.
Principle of enzyme immunoassay (EIA).
Absorbance of -the colored end product is directly proportional to enzyme activity.
Competitive enzyme immunoassay (EIA) mechanism.
Labeled antigen competes with unlabeled patient antigen for binding sites on antibody molecules.
Relationship between enzyme activity and analyte concentration in competitive enzyme immunoassay (EIA).
Enzyme activity is inversely proportional to analyte concentration.
Applications of competitive enzyme immunoassay (EIA).
Used for measuring small, pure antigens such as drugs and hormones.
Mechanism of non-competitive enzyme immunoassay (EIA).
Solid phase antigen, antibody (sample), enzyme-labeled anti-Ig, and substrate are used to measure antibody concentration.
Measurement in non-competitive enzyme immunoassay (EIA).
The amount of color, fluorescence, or luminescence is directly proportional to antibody concentration.
Solid-phase support used in non-competitive enzyme immunoassay (EIA).
Microtiter plates, nitrocellulose membranes, magnetic, or latex plastic beads.
Applications of non-competitive enzyme immunoassay (EIA).
Used for measuring antibody production against infectious agents and for autoantibody testing.
Technique that uses two antibodies, one attached to a solid phase and the other as a label to capture a specific antigen.
Sandwich/Double Ab/Antigen capture technique (Heterogeneous assay).
Type of assay used in ELISA where an antibody is attached to a solid phase, serum with target antigen is added, and labeled AHG is used.
Double Antibody Technique (Sandwich ELISA).
Type of assay in ELISA that is homogeneous, simple to perform, and used for determining low-molecular-weight analytes.
EMIT (Enzyme Multiplied Immunoassay Technique).
Advantages of ELISA, especially in terms of cost and automation.
Cheap, long shelf life, easily adapted to automation, sensitive with inexpensive equipment.
Disadvantages of ELISA related to natural inhibitors and protein binding.
Natural inhibitors, size of enzyme label, nonspecific protein binding.
Immunoassay performed in a plastic cartridge with a membrane that enhances speed and sensitivity of ELISA reactions.
Rapid Immunoassays.
Technique where antigen or antibody migrates through a strip and forms a colored line at the detection zone in the presence of the target.
Immunochromatography.
Type of immunoassay that involves emission of light caused by a chemical reaction with reagents like luminol or acridinium esters.
Chemiluminescent Immunoassay.
Immunoassay that uses chemiluminescent molecules to emit light energy as labels.
Chemiluminescent Immunoassay (CLIA).
Examples of chemiluminescent molecules used in CLIA.
Luminol, Acridinium phosphate ester.
Advantage of chemiluminescent immunoassay due to its light emission detection and stable reagents.
Excellent sensitivity, stable reagents, inexpensive, faster turnaround time.
Type of assay where enzyme activity is inhibited by preventing interaction with the chromogenic substrate.
Enzyme Inhibition.
Type of indirect ELISA where antigen is attached to a well, and patient serum is tested for antibodies.
Indirect ELISA.
Result of colorimetric reaction in indirect ELISA when enzyme-linked antibody binds to antigen.
Color indicates the presence of antibody, directly proportional to antigen-antibody reaction.
Immunoassay that uses colloidal particles like gold or silver as labels.
Sol Particle Immunoassay.
Most commonly used colloidal particle in sol particle immunoassay due to its characteristic magenta-colored product.
Gold.
Colloidal particle used in sol particle immunoassay when gold is too expensive.
Silver.
Type of sol particle immunoassay where one type of particle is used as the label.
Homogeneous Sol Particle Immunoassay.
Type of sol particle immunoassay that uses a mixture of particles and requires separation prior to measurement.
Heterogeneous Sol Particle Immunoassay.
