Transcript Lab.-2-Labeled-Immunoassay-1

LABELED IMMUNOASSAYS
Lab. 2
Dr. M. Alzaharna 2015
IMMUNOASSAY
• An immunoassay is a test that uses antibody
and antigen complexes as a mean of generating a
measurable result
• Immuno refers to an immune response that causes the
body to generate antibodies,
• And assay refers to a test
• An antibody-antigen complex is also known as an immunocomplex
• The assay takes advantage na fo gnidnib cificeps eht fo
negitna sti ot ydobitna
• The antibodies used must have a high affinity for the
antigen
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IMMUNOASSAYS
• Immunoassays derive their unique specificity, sensitivity,
and flexibility from three important properties of
antibodies:
• Their ability to bind to an extremely wide range of natural
and man-made chemicals, biomolecules, cells, and viruses
• Exceptional specificity for the substance to which each
antibody binds.
• The strength of the binding between an antibody and its
target
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Immunoassay
• Both the presence of antigen or antibody can be measured
• For measuring hormones such as insulin, the insulin acts as
the antigen
• When detecting infection the presence of antibody against the
pathogen is measured
• For numerical results the response of the object being measured
must be compared to standards of a known concentration
• This is usually done through the plotting of a standard curve on a
graph paper, and then the quantity of the unknown is found from
the curve
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TYPES OF TESTS
• Many methods of varying sophistication are used for
immunodiagnostic studies
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TYPES OF TESTS
Name of Test
Observable Reaction
Visible Change
Clumping
Agglutination
Particulate antigen reacts with corresponding
antibody; antigen may be in form of RBCs
(hemagglutination, latex, or charcoal coated
with antigen).
Precipitation
Soluble antigen reacts with corresponding
antibody
Precipitates
Complement fixation
(CF)
Competition between two antigen-antibody
systems (test and indicator systems)
Complement activation,
hemolysis
Immunofluorescence
Fluorescent-tagged antibody reacts with antigen- Visible microscopic
antibody complex in the presence of
fluorescence
ultraviolet light.
Enzyme-linked
immunosorbent assay
(ELISA)
Indirect EIA for quantification of an antigen or
antibody enzyme and substrate
Color change indicates
enzyme substrate
reaction.
Immunoblot (eg,
Western blot [WB])
Electrophoresis separation of antigen subspecies
Detection of antibodies of
specific mobility
Measures either antigen or antibody in solution
through the scattering of a light beam;
antibody reagent used to detect antigen IgA,
IgG, IgM; concurrent controls are run to
establish amount of background scatter in
reagents and test samples.
Light scatter
proportionately
increases as numbered
size of immune
complexes increases.
Rate nephelometry
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Rate nephelometry
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Antigen-antibody interaction
• Antigens are substances that stimulate and subsequently react
with the products of an immune response. They may be:
• Enzymes,
• Toxins,
• Microorganisms (eg, bacterial, viral, parasitic, fungal),
• Tumors,
• Autoimmune factors
• Antibodies are proteins produced by the body's immune
system in response to an antigen or antigens.
• The antigen-antibody response is the body's natural defense
against invading organisms
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ANTIBODY STRUCTURE
Antigen
binding
site
V
V
V
Light
Chain
Antigen
binding
site
V
SS
SS
Heavy Chains
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Light
Chain
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ANTIGEN BINDING
Antigen 1
Antigen 3
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HISTORY
•
In the year 1959, Drs. Rosalyn
Yalow & Soloman Berson invented
the radioimmunoassay (RIA)
•
Applied the use of radioisotopes in
the measurement of insulin
•
The RIA is the predecessor of
modern immunoassays
Dr. Rosalyn Yalow became the first
female to win a Nobel Prize with
her work on the radioimmunoassay
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CURRENT SITUATION
• There are now hundreds of immunoassays for
dozens of analytes including:
• Hormones
• Tumor markers
• Drugs
• Cardiac markers
• Antibodies
• Covering the fields of:
• Endocrinology
• Oncology
• Hematology
• Toxicology
• Immunology
• Infectious diseases
• Developments in antibodies, labels, and automation have
resulted in highly specific and sensitive assays
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Constituents of a labeled immunoassay
•
For detection of an analyte, the following are usually a part
of the assay:
1. Labeled analytes
2. Specific antibody
3. Standards or calibrators
4. A method to separate the bound from
free components
5. A method for detection of the label
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1- labeled analytes
• A labeled analyte is used to detect whether or not specific binding
has taken place
• The label used in immunoassay:
• Must not alter the reactivity of the molecule
• And it should remain stable for the shelf life of the reagent
• Labels attached to analytes and antibodies can be:
• Radioactive
• Usually iodine-125 (radioimmunoassay),
• Enzymes
• Such as alkaline phosphatase and horseradish peroxidase, (enzyme
immunoassay or enzyme-linked immunosorbent assay [ELISA]),
• Chemiluminescent
• e.g. Acridinium ester
• Fluorescent
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• e.g. Fluorscein
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Methods of coupling indicator labels to antigen or
antibody
• The methods include:
A. Binding to amino acids
• The radioactive isotope iodine is
covalently linked to tyrosine
residues present on antibodies and
most antigens
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Methods of coupling indicator labels to antigen or antibody
B. Using glutaraldhyde
• It is a bifunctional reagent that
covalently cross links two
aminoacids together, reacts
with amine groups
• Fluorochromes or enzymes
may be coupled to antigens or
antibodies using glutaraldhyde
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Methods of coupling indicator labels to antigen or
antibody
C. Biotin-streptavidin system
• Biotin is a vitamin that can bind tightly to either avidin or
streptavidin (proteins)
• The natural attraction of biotin to these two proteins is a
property that has been exploited to facilitate coupling of
indicator molecules to antigens or antibodies
• At the end of the assay, a conjugate of streptavidin linked to a
signal-generating substance is added
• Examples of suitable conjugates are streptavidin-alkaline
phosphatase, streptavidin-HRP, streptavidin-125I,
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streptavidin fluorescein
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THE BIOTIN-STREPTAVIDIN INDICATOR LABEL
SYSTEM
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2- Production of Antibodies
• The production of antibodies is an important process in the use of
immunoassays because it is the antibody-antigen complexes that
form the basic
• Antibodies can be called depending upon the technique used to
produce them either:
a) Monoclonal or
b) Polyclonal
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A) POLYCLONAL ANTIBODIES
• May be produced in mammals such as rabbits or
sheep
• When a foreign substance enters the body, it stimulates the
immune system to produce antibodies to the substance
• Using this natural reaction, an analyte in as pure form as possible
is injected into the animal stimulating the production of
antibodies
• Antiserum usually contains a mixture of antibodies that
recognize and bind to the same antigen, but they may attach to 20
different epitopes
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A) POLYCLONAL ANTIBODIES
Ag
Multivalent Antigen
complex
Ag
Polyclonal Antibodies
Antibody-Antigen
complex
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B) MONOCLONAL ANTIBODIES
• Monoclonal antibodies production result in very specific
antibodies that bind only to one antigen epitope, which in turn
reduces the occurrence of false positives in the immunoassay
Ag
Multivalent Antigen
complex
Ag
Monoclonal Antibodies
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Antibody-Antigen
complex
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b) Monoclonal Antibodies
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3- Standards or Calibrators
•
Calibrators are solutions with known
values that establish the relationship
between the amount of signal
produced in the assay and analyte
concentration
• By running a set of calibrators, a calibration curve is set up in the
instrument’s software and correlates certain values of signal to
known analyte concentrations
• By comparing levels of signal produced by patient samples to this
calibration curve, a patient analyte concentration value, or result,
can be determined
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4- Separation Methods
• In most assays, once the reaction between antigen and antibody has taken
place, there must be a way of separating reacted from unreacted analyte
• This can be accomplished by several different means
• Unreacted analyte can be removed by:
• Adsorption on particles such as dextran-coated charcoal
• These adsorb out the smaller unbound molecules, which are then
separated from bound molecules by centrifugation or filtration
• The amount of label remaining in the supernatant provides an indirect
measure of analyte present in the patient's sample
• Another means of separation involves precipitation of antigen-antibody
complexes
• Complexes can be precipitated by adding concentrated solutions of 25
ammonium sulfate, or ethanol
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• Currently, most immunoassays use a solid-phase stage for
separation
• Numerous substances, such as polystyrene test tubes, microtiter
plates are used for this purpose
• Antigen or antibody is attached by physical adsorption, and when
specific binding takes place, complexes remain attached to the
solid phase
• This provides a simple way to separate bound and free reactants
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5- Methods for Detection of Label
• The last step common for all immunoassays is detection of
the labeled analyte
• The method depends on the label;
• e.g. 125I is easily detected by a γ-counter
• Enzymes are generally used to produce coloured products
from colourless substrates that can be determined easily
using a spectrophotometer
• Automated plate readers are commercially available
which make reading large numbers of samples
relatively easy
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Quality control in immunoassays
• It is essential that quality control procedures be established
• This is done to limit random errors, such as
• Temperature fluctuations
• Minor changes in the concentration of reagents
• And changes in detector efficiency
• A negative control and a positive control should be run
• This serves as a check on the quality of the reagents to make sure
that the label is readily detectable under current testing conditions
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Methodological principles
1. Competitive immunoassays
2. Noncompetitive immunoassays
3. Heterogeneous immunoassays
4. Homogeneous immunoassays
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1- Competitive Immunoassays
• Based on competition between the analyte in the
sample and a labeled analyte for a limited mount
of antibody
Coating
Incubation
S
L
L
L
L
Product
measurement
P
L
immobilisation surface
Specific Ab
Ag
Enzym.
reaction
L
antigen- enzyme conjugate
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1- Competitive immunoassays
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2- Noncompetitive immunoassays
• Noncompetitive immunoassays (sandwich) generally provide the
highest level of assay sensitivity and specificity
• The reaction mixture typically includes an excess of bound antibody, so that
all analyte is bound
• The amount of antibody-antigen complex is then measured to determine the
amount of analyte present in the sample
• The labeled antibody, is directly proportional to the amount of antigen
present in the sample
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Homogeneous Versus Heterogeneous
Immunoassays
3- Heterogeneous immunoassay
• Methods that require separation of bound ab-ag* complex
4- Homogeneous immunoassay
• Those that do not require separation
• Homogeneous methods have been generally applied to the
measurement of small analytes such as abused and
therapeutic drugs
• Since homogeneous methods do not require the separation of
the bound ab-ag* from the free ag*, they are generally much
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easier and faster to perform
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Types of Immunoassays (Heterogeneous)
• Within the categories of competitive, noncompetitive,
homogenous, and heterogeneous, there are specific types,
which include:
• Radioimmunoassays
(RIAS)
• Utilize a radioactive label
(usually 125I, 3H or 14C),
which emits radiation that
can be measured with a beta
or gamma counter
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Types of immunoassays (Heterogeneous)
• Enzyme Linked
Immunosorbant Assay
(ELISA):
• Direct, sandwich and
competitive
• Reaction components are
absorbed or bound to the
surface of a solid phase,
commonly a well of a
microtiter plate
• Absorbance is measured
using a micro-plate
reader
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Types of immunoassays (homogeneous)
• Enzyme multiplied
immunoassay (EMIT)
• The drug in the sample
and the drug labeled with
G6PD compete for
antibody binding sites
• Binding inhibits enzyme
activity, while free
enzyme remains active to
interact with the
substrate
• Enzyme
activity/absorbance is
directly proportional to
drug concentration.
Substrate reacting
with enzyme
Substrate
Enzyme with
drug attached
Drug blocking drug
attached to enzyme
Antibody
Drug
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• Qualitative
• Single point calibration at a specific cutoff
• Results are either ‘positive’ or ‘negative’ (i.E. Above or
below the cutoff)
• Quantitative
• Provides numeric results that are an estimate the
analyte concentration based on the measurement
standards
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Anti HIV1/2 TEST KIT
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HUMAN IMMUNODEFICIENCY
VIRUS






The causative agent of acquired
immunodeficiency syndrome (AIDS).
Belong to the lentivirus subfamily of the
retroviridae family.
Enveloped, icosahedral, RNA containing
particles.
Have envelope glycoproteins: gp120 and gp41.
Infect immune system (T helper lymphocyte;
CD4).
Incubation period: 3 – 5 years.
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TRANSMISSION

Sexual contact (primarily).

Sharing contaminated intravenous needles.

Infected mother to her baby, either across the placenta,
at birth, or via breast milk.

Blood transfusions and tissue transplantation.
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SIGNS AND SYMPTOMS
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TEST KIT INTENDED USE
• SD HIV-1/2 ELISA 3.0 kit is sandwich ELISA for the qualitative
detection of antibodies to all isotypes(IgG, IgM, IgA) specific to
HIV-1 including subtype-o and HIV-2 simultaneously in human
serum or plasma
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PRINCIPLE
• SD HIV-1/2 ELISA contains a microplate, which is pre-coated with
recombinant HIV ½ antigens (gb41, P24, gp36) on well. During first
incubation, anti-HIV in patient serum is bound to the recombinant
HIV1/2 antigens. Following this incubation, all unbound materials
are removed by washing. The recombinant HIV1/2 antigensenzyme conjugate (gp41, P24, gp36) is bound to antiHIV1/2,by
performing a sandwich.
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PROCEDURE
1.
Prepare the strip wells for negative control, positive control and sample
2.
Pipette 100 ul of sample diluent to each well
3.
Add 50 ul of positive and negative controls.
4.
Cover the microplate and mix well.
5.
Incubate the wells for 30 min at 37 C
6.
Wash the wells 5 times with 350 of diluted washing solution.
7.
Pipette 100 ul of enzyme conjugate to each well
8.
Cover the wells
9.
Incubate at 37 C for 30 min.
10. Wash 5 times.
11. Add 100 ul of mixed substrates (50ul A + 50ul B)
12. Incubate for 10 min
13. Pipette 100 ul of stopping solution
14. Read the absorbance at 450 nm
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CALCULATION OF RESULTS
1.
Calculate the mean value of the negative controls, then calculate the
cut-off value by adding 0.3.
A(neg) + 0.3 = cut-off
Abs. of negative controls = 0.193 , 0.167
Abs. of positive controls=1.216, 1.260
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VALIDATION REQUIREMENT AND
QUALITY CONTROL
The individual values of the absorbance for the control sera are used to
calculate the mean value if
0.010<A(neg)<0.200
A (pos)>1.000
If thes e specifications are not met, the test must be repeated.
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INTERPRETATION OF RESULTS
• A sample < cut-off  anti-HIV1/2 negative
• A sample > or equals cut-off  anti-HIV1/2 positive
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