Fluorescent Immunoassays (FIA) Immunology Review 394
METHOD
Direct fluorescent
antibody (DFA)
staining
Indirect fluorescent
antibody (IFA)
staining
Fluorescence
polarization
immunoassay
(FPIA)
EXAMPLES OF ANALYTES
Bacterial, viral antigens
Fluorescent antinuclear
antibody (FANA), fluorescent
treponemal antibody (FTA)
Therapeutic drugs, hormones
PRINCIPLE
Specimen on glass slide overlaid with fluorescein-
labeled ab. If corresponding ag present, labeled
ab binds. Fluorescence observed with fluorescent
microscope.
Reagent ag on glass slide overlaid with patient
serum. If corresponding ab present in serum, at-
taches to ag. When fluorescein-labeled antihuman
globulin added, attaches to ab. Fluorescence
observed with fluorescent microscope.
Labeled ag competes with ag in specimen for sites
on reagent ab. Free labeled ag rotates rapidly,
emits little polarized light. Bound labeled ag ro-
tates more slowly, emits more polarized light.
Amount of polarized light is inversely proportional
to concentration of ag in specimen.
OTHER
Detects ags. Fluorescent
labels: fluorescein isothio-
cyanate or rhodamine B
isothiocyanate.
“Sandwich technique.”
Detects abs in serum.
Competitive. Homoge-
neous. Automated.
