Quiz 1 Review ( Immunoassays) Flashcards
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
What is the zone of equivalence in a precipitation test?
The point in an antigen-antibody reaction where the number of antigen site and
antibodies are approximately equal.
Optimum precipitation occurs in the zone of equivalence
What are examples of passive precipitation methods in gel?
Double diffusion (Ouchterlony)
Radial immunodiffusion (RID)
What is a pattern of identity in the Ouchterlony technique?
Single, continuous precipitin arc forms meaning antigens in the specimen and in the control are identical.
What is a pattern of identity in the Ouchterlony technique?
Single, continuous precipitin arc forms meaning antigens in the specimen and in the control are identical.
When is immunofixation electrophoresis (IFE) used?
To identify immunoglobulins and Bence Jones protein.
What technique measures protein based on light scatter by immune
complexes?
Nephelometry
What type of specimen could cause inaccurate results in the above
method?
Lipemic specimens would cause light scattering.
