LABELED IMMUNOASSAYS

 LABEL: used to detect Ag-Ab activity diminishes when the Ag and Ab

complex binding occurs
molecules which aid in the identification
of Ag-Ab complex MAJOR FORMATS FOR LABELED
Indicator labels: radio isotopes ASSAYS
(mostly used), enzymes, chloroforms,
and chemiluminesence  Competitive assay
Design for Ag and Ab that may be small - used LABELED antigen and
in size, this is useful when the Ag and UNLABELED antigen which compete
Ab is present in very low concentration for the binding site of an antibody
The presence of Ag and Ab is determine - used to detect antigen in the sample
indirectly by using a labeled reactant to Ex: hormones
detect whether or not specific binding - the amount of bound antigen is
taken place inversely proportional to the antigen
The substance that are being measuring concentration in the sample
is the ANALYTE - the one that we are If there is a great amount or
testing and measuring in the lab concentration of labeled Ag that means
EX: bacterial antigen, hormones, drugs, the Ag concentration is low (inverse
tumor markers, and immunoglobulins relationship)
(IgG, IgM, IgA, IgD, IgE)  Non-competitive assay
This analytes are bound by molecules - antibody and antigen reacts without
that reacts specifically competitive molecules
One reactants, either the Ag or Ab is - uses to detect Ag and Ab in the
labeled with a marker, the amount of sample, both Ab and Ag could be
binding can be monitored measured in non-competitive assay and
Label can be either attached or labeled it is directly proportional reaction
with a marker
Labeled immunoassays - made possible
rapid quantitative measurement of many
entities such as viral antigens, in patients
with infected with HIV
 More sensitive than precipitation
and agglutination

TERMINOLOGIES
 Solid Phase - this are the weils or
microplates where the Ag and Ab is
fixed
Ex: glass beads, cellulose
membranes, microplates,
polystyrene test tube
 Heterogenous - this requires
washing after reagent, and sample
are mixed to remove unbound
reactant
- There is a separation step
 Homogenous - it doesn’t require
washing, the reagent and sample are
mixed
- There is no separation step, the enzyme

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 LABELED IMMUNOASSAYS
(a) the amount of labeled antigen bound - There is a low quantity self-
is inversely proportional to the radioactivity that can be easily measured
concentration of the labeled antigen It can also be competitive and non-
If there is a high amount or competitive immuno-assay
large concentration of labeled  Measures gamma rays produced by
antigen bound to the antibody, radio isotopes
the concentration of the labeled  Instrument: Scintillation counter -
antigen is low used for measuring ionizing
The more labeled detected, radiations or radioactivity
there is less antigen present in
the patient 2 TYPES OF SCINTILLATION
(b) the antibody often called “capture COUNTER
antibody” Crystal Scintillation Counter -
Antibody is absorbed to a measures gamma rays
solid phase, the unknown patient Liquid Scintillation Counter -
antigen is then allowed to react measures beta rays
with Ab and captured by it
After washing, to remove the RIA - the most sensitive type of
unbound antigen a second immunoassay
antibody with a label is then
added to the reaction. Disadvantages:
The amount of labeled  Health hazard - involves with
measured is directly exposure radioactive substances
proportional to the amount of  Expensive - more difficult and
patient antigen expensive to maintain a license and
to comply regulations
Non competitive (no labeled  Difficult to operate - it needs
Ag, we add labeled Ab to the proper/intensive training, the staff
reaction) second antibody is must be skilled and expert to
used operate in this type of assay
The reaction is directly  Disposal problems - there is a need
proportional for expensive equipment that has
cost laboratory to explore other
TYPES OF IMMUNOASSAYS techniques

RADIOIMMUNOASSAY
(RIA)
 1st type to developed by scientist:
Yallow and Berson
 Label: 125I; 3H tritiated
hydrogen, 131 iodine
125I (mostly used radio isotope in
RIA)
- It has a half length of 60 days, it has a
higher counting rate than 3H the total
counting time is less, mas mabilis ang
reaction with the use of 125I RADIOIMMUNOSORBENT TEST
- Easily incorporated in protein (RIST)
molecules and emits gamma radiation  For measurement of total IgE
(detected by a gamma counter)

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 LABELED IMMUNOASSAYS
RADIOALLERGOSORBENT TEST
(RAST) NON-COMPETITIVE ENZYME-
 For measurement of specific IgE LINED IMMUNOSORBENT ASSAY
(ELISA)
ENZYME IMMUNOASSAY (EIA)  INDIRECT ELISA - the enzyme
 Label: horse radish peroxidase labeled reagent does not participate
(most commonly used enzyme), in the initial Ag and Ab binding
ALP, glucose oxidase, beta reaction
galactosidase, G6PD  Amount of enzyme is directly
- most widely used because it is cheap, it proportional to the amount of the
is easily adapted to automation, no test substance
health hazard, and it is easy to perform, - opposite of COMPETITIVE ELISA
we are not dealing with radioactive - when there high enzyme activity, the
substances concentration of Ag or Ab is high
Like RIA, it can be competitive or non
competitive CAPTURE ASSAYS/SANDWICH
 Instrument: spectrophotometer  Enzymatic activity is directly
proportional to the amount of
COMPETITIVE ENZYME-LINKED antigen in the test sample
IMMUNOSORBENT ASSAY (ELISA) - if there is high activity of enzyme,
 Competitive assays based on means there is a high concentration of
principles of RIA Ag present in the patient
Same principle with RIA, but it is just - the antigen captured must have
competitive multiple epitopes, multiple antigenic
If the concentration of enzyme is high determinants
means the test cells substance is low - prone to hook effect (when there is
If there is low enzyme activity the excess of Ag, there is Ag excess (POST
concentration of the test substance is ZONE REACTION)
high - When there is antigen excess, the
An enzyme labeled reagent are used to majority of the binding sites are field,
detect Ag or Ab, the enzyme that is used the remainder of the patient Ag has no
should be stable, specific and cannot place to bind
bind to Ag or Ab independently - When the hook effect happens, this
Procedure: a colorless substrate is result in unexpected fall in amount of
metabolized by the enzyme into a measured analyte when an extremely
colored compound high concentration is present
The intensity of the color is directly
proportional to the amount of the
enzyme present
 Enzyme activity is inversely
proportional to the concentration of
the test substance

IMMUNOENZYMETRIC
Color intensity is directly proportional IMMUNOASSAY
to the enzyme concentration or enzyme  Enzyme is directly proportional to
activity but the enzyme concentration is antigen concentration
inversely proportional to the Ag
concentration or Ab concentration

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 LABELED IMMUNOASSAYS
- kung ano ang activity ng enzyme, that pallidum bacteria that is a causative
means same din ang concentration ng agent of sysphilis
Ag
High/high or low/low BIOTIN-AVIDIN
IMMUNOFLUORESCENCE
FLUORESCENT IMMUNOASSAY INDIRECT ASSAY
- the Ab are labeled with fluorescent dye - the detection system is modified by
Label: fluorochromes/fluorophores using a biotin labeled Ab followed by
(fluoresciin isothiocynate (this is green Avidin labeled chloroform
in color) tetramethyehodamine
isothiocynate (red in color) b-napthyl
trifluoroacetate (lucifer/yellow color) - this dyes or chloroform they absorbed
light, after adding them we will
Instrument: fluorescence observed under fluorescence microscope
microscope/flurometer
Flourochromes - absored light and get
excited (principle)
Can be direct (detects Ag) or indirect
(detects Ab)
- the atoms emits light of longer
wavelength and lower energy during
transition to the ground from excited
state
- this type of assay is extremely specific
and very sensitive INHIBITION FLUORESCENT
ASSAY
DIRECT FLUORESCENT - blocking test in which Ag is 1st
IMMUNOASSAY exposed to unlabeled Ab
For immunohistochemistry; detects - after exposing to unlabeled Ab, it is
antigen/epitope - the unknown antigen exposed to another labeled Ab and it is
is fixed to a microscope slide finally washed and examine
- The fluorescent labeled antibody are If the labeled and unlabeled antibodies
added directly to the unknown antigen are both homologous to the antigen,
- After that, the slide is ready using a there should be NO fluorescence
fluorescent microscope - the positive result here should be NO
FLUORESCENCE
INDIRECT FLUORESCENT - this result confirms the specificity of
IMMUNOASSAY the fluorescent immunoassay
The same principle with INDIRECT - the Ab in an unknown serum can also
ELISA be detected and identified by this
inhibition test
Ex: FANA (flourescent antinuclear
antibodies) FLUORESCENCE POLARIZATION
IMMUNOASSAY (FPIA)
Based on the change in polarization of
FTA-ABS (flourescent trepanomal fluorescent light emitted from a labeled
antibody absoprtion test) molecule when it is bound by antibody
- a blood test that checks for the
presence of Ab to trepanoma

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