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IMMUNOASS
AY
CHARACTERISTICS OF LABELED
ASSAY
Competitive
COMPETITIVE
IMMUNOASSAY
all the reactants are mixed together
simultaneously, and labeled antigen competes with
unlabeled patient antigen for a limited number of
antibody-binding sites
CHARACTERISTICS OF LABELED
ASSAY
Competitive
NONCOMPETITIVE
IMMUNOASSAY
Antibody(often called capture antibody) is first
passively absorbed to a solid phase
A second antibody bearing a label is added and
binds wherever there is patient antigen
CHARACTERISTICS OF LABELED
ASSAY
CHARACTERISTICS OF LABELED
ASSAY
Antibodies
CHARACTERISTICS OF LABELED
ASSAY
o
Standard or Calibrators
CHARACTERISTICS OF LABELED
ASSAY
Separation
method
CHARACTERISTICS OF LABELED
ASSAY
Detection
of the label
CHARACTERISTICS OF LABELED
ASSAY
Quality
Control
RADIOIMMUNOAASAY
RADIOIMMUNOASSAY
RADIOIMMUNOASSAY
PRINCIPLE OF RIA
Labeled antigen
competes with
patient Ag for a
limited number of
binding sites on
solid-phase
antibody
PRINCIPLE OF RIA
A.
B.
ENZYME
IMMUNOAASAY
ENZYME IMMUNOASSAY
HETEROGENEOUS ENZYME
IMMUNOASSAY
HETEROGENEOUS ENZYME
IMMUNOASSAY
Noncompetitive EIA (enzyme-linked
immunosorbent assay/ELISA)
One of the most frequently used immunoassays in
the Clinical Lab
This type of assay has been used to measure
antibody production to infectious agents that are
difficult to isolate in the laboratory and has been
used for autoantibody testing.
Noncompetitive EIA
HOMOGENEOUS ENZYME
IMMUNOASSAY
HOMOGENEOUS ENZYME
IMMUNOASSAY
1.
2.
3.
4.
FLUORESCENT
IMMUNOAASAY
FLUORESCENT IMMUNOASSAY
FLUORESCENT IMMUNOASSAY
Fluorophores- typically organuc molecules with
a ring structure, and each has a characteristic
optimum absorption range.
Fluorescein and Rhodamine- compounds most
often used.
Fluorescein- absorbs maximally at 490 to 495
nm and emits a green color at 517 to 520 nm
Tetramethylrhodamine- absorbs at 550 nm
and emits red light at 580 to 585 nm
IFA( Immunodfluorescent assay)
-a term restricted to qualitative observations
involving the use of fluorescence microscope.
TYPES:
Direct IFA
antibody that is conjugated w/ fluorescent tag is
added directly to unknown antigen that is fixed
to microscope slide. -best suited to and antigen
detection in tissue or body fluids.
Examples of antigen detected by this method:
Legionella pneumophila, Pneumocystis
carinii,Chlamydia trachomatis, Respiratory
syncytial virus
TYPES:
Indirect IFA
-involves two step: Incubation of patient serum with
a unknown antigen attached to solid phase. The
slide is washed, and then an antihuman
immunoglobulin containing a fluorescent tag is
added. This combines with the first antibody to form
a sandwich, which localizes the fluorescence.
-result in increased staining, because multiple
molecules can bind to each primary molecule, thus
making this a more sensitive technique.
-useful in antibody identification and have been
used to detect treponema, antinuclear, chlamydial,
and taxoplasma antibodies.
TYPES:
OTHER TYPES:
OTHER TYPES:
Fluorescence polarization immunoassay (FPIA)
most popular technique.
based on the change in polarization of fluorescent
light emitted from a labeled molecule when it is
bound by antibody.
labeled antigen compete with unlabeled antigen in
the patient sample for a limited number of antibody
binding sites.
the more antigen present in the patient sample, the
less the fluorescence
labeled antigen bound and the less polarization that
will detected.
CHEMILUMINESCENT
IMMUNOAASAY
CHEMILUMINISCENT IMMUNOASSAY
PRICIPLE:
Chemiluminescence
employed to follow antigen-antibody
is the emission of light caused by chemical
reaction, typically an oxidation reaction,
producing an excited molecule that decays back
to its original ground state.
This type of labeling can be used for
heterogeneous and homogeneous.
CHEMILUMINISCENT IMMUNOASSAY
Luminol,acridinium, ester, ruthnium
derivatives and nitrophenyl- most common
substances used that are capable of
chemiluminescence.
Acridinium ester emit a quick burst or flash of
light.
Luminol and dioxetane-the light remains for a
longer time.
PRINCIPLE: