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LABELED

IMMUNOASS
AY

CHARACTERISTICS OF LABELED
ASSAY
Competitive

versus Noncompetitive Assay

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

The amount of bound label is inversely


proportional to the concentration of the labeled
antigen

CHARACTERISTICS OF LABELED
ASSAY
Competitive

versus Noncompetitive Assay

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

The amount of labeled measured is directly


proportional to the amount of patient antigen.

CHARACTERISTICS OF LABELED
ASSAY

In both types of assays, the label must not alter


the reactivity of the molecule, and it should
remain stable for the shelf life of the reagent.
Radioactivity, enzymes, fluorescent compounds,
and chemiluminescent substances have all been
used as labels.

CHARACTERISTICS OF LABELED
ASSAY
Antibodies

Antibodies used in immunoassays must be very


specific and have a high affinity for the antigen in
question.

CHARACTERISTICS OF LABELED
ASSAY
o

Standard or Calibrators

are unlabeled analytes that are made up in a


known concentrations of the substance to be
measured.
are used to establish a relationship between the
labeled analyte measured and any unlabeled
analyte might be present in patient specimens.

CHARACTERISTICS OF LABELED
ASSAY
Separation

method

Bound and unbound label are separated by using a


solid phase surface, such as glass beads, cellulose
membranes, polystyrene test tubes, or microtiter
plates, to attach either antigen or antiboby.

CHARACTERISTICS OF LABELED
ASSAY
Detection

of the label

The last step common to all immunoassay.


For radioimmunoassay, this involves a system for
counting radioactivity, while for other labels such
as enzymes, fluorescence, or chemiluminescence,
typically a change in absorbance in a subs is
measured be spectrophotometer.

CHARACTERISTICS OF LABELED
ASSAY
Quality

Control

Any readings indicative of label in the blank are


known as backgrounds.
If the background is too high, wash steps need to be
made more efficient.
A negative control and a high and a low positive
control should be run in addition.
All controls and the patient samples are usually run
in duplicate.
If any controls are out of range, test values should be
reported.

RADIOIMMUNOAASAY

RADIOIMMUNOASSAY

First type of immunoassay developed by Yalow


and Berson in the late 1950s.
It was used to determine the level of insulinanti-insulin complexes in diabetic patients.
Several radioactive labels, including 131I, 125I,
3
H, have been used .
125
I is the most popular.

RADIOIMMUNOASSAY

The original technique was based on competition


between labeled and unlabeled antigen for a
limited number of antibody-binding sites.
The more analyte that is present in the patient
sample, the lower the amount of radioactivity
that is detected.

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.

Very little patient


Ag is present,
making
radioactivity of the
solid phase high.
More patient Ag is
present, & the
radioactivity of the
solid phase is
reduced in
proportion to the
amount of the
patient Ag bound

ENZYME
IMMUNOAASAY

ENZYME IMMUNOASSAY

Enzymes can be used as labels in much the same


manner as radioactivity.
Enzymes used as labels for immunoassay are typically
chosen according to the number of substrate molecules
converted per molecule of enzyme, ease and speed of
detection, and stability
Enzyme assays are classified as either
Heterogeneous or Homogeneous

HETEROGENEOUS ENZYME
IMMUNOASSAY

Heterogeneous enzyme immunoassay require a


step to physically separate free from bound analyte.
Competitive EIA based on the principles of RIA.
Enzyme activity is inversely proportional to the
concentration of the test substance.
This method is typically used for measuring small
antigens that are relatively pure, such as insulinn
and estrogen.

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

Patient antibody is incubated with solid-phase Ag.


After a wash step, enzyme-labeled antiimmunoglobulin is added. This will bind to the
patient Ab on solid Phase

A second wash step is performed to remove any


unbound anti-immunoglobulin, and substrate for the
enzyme is added

Color development is directly proportional to the


amount of patient antibody present.

HOMOGENEOUS ENZYME
IMMUNOASSAY

Require no separation step.


They are based on the principle that enzyme
activity changes as specific antigen- antibody
binding occurs.
When antibody binds to enzyme-labeled antigen,
steric hindrance results in a loss in enzyme
activity.
Example: the Enzyme-multiplied
Immunoassay Technique(EMIT) developed by
Syva Corporation

HOMOGENEOUS ENZYME
IMMUNOASSAY

1.
2.
3.
4.

The sensitivity is determined by the ff:


Detectability of enzymatic activity
Change in that activity when antibody binds to
antigen
Strength of the antibodys binding
Susceptibility of the assay to interference from
endogenous enzyme activity, cross reacting
antigens, or enzyme inhibitors.

FLUORESCENT
IMMUNOAASAY

FLUORESCENT IMMUNOASSAY

1941, Albert Coons demonstrated that antibodies


could be labeled with molecules that fluoresce
Called Fluorophores or Fluorochromes
they can absorb energy from an incident light source
and convert that energy into light of a longer
wavelength.

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:

A.Direct fluorescent assay


B.Indirect fluorescent assay

OTHER TYPES:

Quantitative fluorescent immunoassay (FIAs)


can be classified as heterogeneous or homogeneous,
corresponding to similar types of enzymes
immunoassay.
Solid-phase heterogeneous fluorescent assay
developed for the identification of antibodies to
nuclear ag, toxoplasma Ag, rubella virus, and other
viruses

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:

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