Quiz 1 Review ( Immunoassays)
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What are immunoassays?
Tests that are based on antigen-antibody reactions
Immunoassays are used to measure what concentrations of analytes?
< 1 μmol/L
The region of the antigen that binds to a complementary antibody is called?
An epitope
The strength of a single antibody-antigen interaction is called?
Affinity
The combined strength of all antibody-antigen interactions is called?
Avidity
What is cross-reactivity?
The ability of an antibody to react with more than one epitope. (i.e. Ab reacts with structurally similar antigens)
Which is more sensitive labeled or unlabeled immunoassays?
Labeled
What types of labels can be used in labeled immunoassays?
Radioactive element Fluorochrome Chemiluminescent compound Enzyme
What are the common chemiluminescent labels?
Luminol Acridinium esters
What are the most common enzyme labels?
Alkaline phosphatase (ALP) Horseradish peroxidase (HPO)
What is the principle of competitive immunoassays?
Unlabeled antigen in the patient sample competes with labeled reagent antigen for limited binding sites on a reagent antibody. (Equal competition) Labeled Ag is inversely proportional to patient analyte concentration. The more unlabeled (patient) antigen in the sample, the less labeled antigen will be bound.
What is the difference between heterogeneous and homogeneous immunoassays?
Heterogeneous immunoassays require a separation step of antigen bound to antibody from unbound antigen. Homogeneous immunoassays do not require a separation step.
What are some examples of competitive, heterogeneous immunoassays?
RIA - radioimmunoassay EIA - enzyme immunoassay FIA - fluorescence immunoassay ELISA - enzyme-linked immunosorbent assay
What are the steps in an ELISA immunoassay to detect an antibody?
• Patient sample is added to a microtiter well containing reagent antigen. If antibody is present in the sample, it will bind to the reagent antigen on the well. • A wash step removes any unbound sample. • Enzyme-labeled antibody is added and will attach to the patient antibody if present. • A wash step removes any unbound labeled antibody. • Enzyme substrate is added.
In a competitive immunoassay, what relationship exists between the unlabeled patient antigen and the bound labeled antigen?
The more unlabeled patient antigen in the sample, the less bound labeled antigen. This will mean less radioactivity, color, fluorescence, chemiluminescence, etc. Inversely related.
What are some examples of competitive, homogeneous immunoassays?
EMIT - enzyme-multiplied immunoassay technique FPIA - fluorescence polarization immunoassay
How do non-competitive immunoassays differ from competitive immunoassays?
Non-competitive immunoassays use an excess of labeled antibody. The concentration of patient antigen is directly proportional to the bound, labeled antibody.
What are some examples of non-competitive immunoassays?
IRMA - immunoradiometric assay IEMA - immunoenzymatic assay
What can cause false-positive reactions with sandwich immunoassays?
Heterophile antibodies HAMA
What can cause false-negative results when using sandwich immunoassays?
The hook effect Occurs when analyte being measured is in very high concentrations. There is insufficient binding sites for all of the antigen in the sample.
How can the hook effect be corrected?
Dilute the sample and rerun.
Which immunoassay has a direct relationship between the concentration of drug in a patient sample and the activity of free labeled drug?
EMIT
In fluorescence polarization immunoassays (FPIAs), how does the light emission of bound labeled antigen differ from that of free labeled antigen?
Bound labeled antigen rotates more slowly and emits more polarized florescence. Free labeled antigen rotates more quickly and emits less polarized florescence. No separation step required
What are some examples of non-labeled immunoassays?
Nephelometry Turbidometry Precipitation methods