Transcript Lab. 3 Labeled Immunoassays

LABELED
IMMUNOASSAYS
PART 2
Enzyme Linked Immunosorbent Assay (ELISA)
Lab. 3
Labeled Immunoassays
• The basic underlying principles of indicator
labeled immunoassays are the same
• There are differences with respect to the detail of
the protocols
• The designation given to each test differs
according to the label used to detect the antigen/
antibody complexes
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Enzyme Linked Immunosorbent Assay
(ELISA)
• One of many assays collectively called enzyme
immunoassays (EIA)
• Can be used to detect both antibody and
antigen
• Very Sensitive (ng & pg/mL),
• Relies on Monoclonal Abs
• An enzyme is used as an indicator molecule
• The enzyme does not provide detection directly
but through the break-down of a substrate
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Enzymes used in ELISA
• Enzymes used as labels for immunoassay are
chosen according to the following criteria:
 Turnover number
 Sensitivity
 Ease and speed of detection
 Stability
 Absence of interfering factors in patient samples
 Availability and cost of enzyme and substrate
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Enzymes used in ELISA
• The most commonly used enzymes are:
 Horseradish peroxidase (HRP) and
 Alkaline phosphatase (AP)
• Each has a high turnover number (rapid conversion of
substrate to a product) resulting in high sensitivity
• Other enzymes have been used as well, but they have not
gained widespread acceptance because of limited
substrate options
• These include beta-galactosidase, acetylcholinesterase and
catalase
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Enzyme Substrates
• A large selection of substrates is available for performing the ELISA
with an HRP or AP conjugate
• Substrates for AP and HRP, depending upon the plate-reading
equipment available and the level of sensitivity required in the ELISA
• An array of, and substrates is available for use with either enzyme
• Chromogenic
Color
• Fluorogenic
Fluoroescence
• Chemiluminescent
spectrophotometer
light
fluorometer
luminometer
• Chemiluminescent and chemifluorescent substrates provide a
stronger signal than Chromogenic substrates
• Steadily gaining in popularity because of their:
• sensitivity (less than 1 pg/ml “10-12”),
• large linear range for detection
• and excellent antibody conservation
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Enzyme Substrates
1. Chromogenic substrates
• A common ELISA substrate for HRP is:
• Tetramethylbenzidine (TMB),
• TMB is oxidized to yield a blue product that is water-soluble and absorbs
light at 650 nm
• The reaction can be halted by addition of acid or another stop reagent
• Using sulfuric acid turns TMB yellow color which may be read at 450 nm
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Enzyme Substrates
• The most common chromogenic substrate for alkaline
phosphatase is:
• p-nitrophenyl phosphate (PNPP)
• PNPP yields a yellow reaction product that is water-soluble and
absorbs light at 405 nm
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Enzyme Substrates
2. Chemiluminescent substrates
• Luminol is one of the most widely used chemiluminescent
reagents and its oxidation by peroxide results in creation of an
excited state product called 3-aminophthalate
• This product decays to a lower energy state by releasing photons
of light
• Using chemiluminescence allows multiple exposures to be
performed to obtain the best image
• Sensitivity: picogram or femtogram level (1.0 × 10-15 grams)
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Enzyme Substrates
• Fluorogenic substartes
• A Fluorogenic Substrate is a nonfluorescent
material that is acted upon by an enzyme to
produce a fluorescent compound
• Different substartes are also available for AP
and HRP enzymes
• p-Hydroxphenylpropionic acid (HPPA) is used
for HRP enzyme
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Linkage of Enzyme
The enzyme label is linked to antibody or analyte
by several means
1. Glutaraldehyde is often used as a crosslinker to join
amino groups of the enzyme and the molecule to be
labeled
2. Maleimide derivatives are also used to attach the
enzyme label
 The heterobifunctional cross-linker
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Linkage of Enzyme
3. Treatment with periodate
• Glycoproteins such as
horseradish peroxidase can
be activated for conjugation
by treatment with periodate
• This provides a mild and
efficient way of generating
reactive aldehyde groups for
subsequent conjugation with
amine- or hydrazidecontaining molecules
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Linkage of Enzyme
4. The use of biotin-
avidin binding
• The secondary Ab is
labeled with biotin
• An enzyme-avidin
conjugate is added
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Coating of Microplate
• A key feature of the solid-phase ELISA is
that antigens or antibodies can be attached
to surfaces easily by passive adsorption
• This process is commonly called coating
• Most proteins adsorb to plastic surfaces,
probably as a result of hydrophobic
interactions between nonpolar protein
substructures and the plastic matrix
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Coating of Microplate
• Since most of proteins' hydrophilic residues are at
the outside and most of the hydrophobic residues
orientated towards the inside
• Partial denaturation of some proteins results in
exposure of hydrophobic regions and ensures
firmer interaction with the plastic
• This can be achieved by exposing proteins to low
pH or mild detergent
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ELISA Protocol
1- Coating antibody or antigen onto the microplate


Dilute the protein to be coated in a buffer such as PBS or
Carbonate-Bicarbonate and add 100 μl of this solution per well
Incubate for 18-20 hours at room temperature or 4°C
Coated with Antibody
when analysing antigen
Coated with Antigen
when analysing antibody
Analyte = antibody
Analyte = antigen
Incubate, wash
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ELISA Protocol
2- Blockage of free binding sites

Block the unoccupied sites on the surface of the well to reduce
the amount of nonspecific binding of proteins [blocking agent (200300 μl/well)]


A variety of blocking buffers ranging from nonfat milk to highly
purified proteins have been used to block unreacted sites
The blocking buffer should improve the sensitivity of the assay by
reducing the background interference
Analyte = antibody
Analyte = antigen
Incubate, wash
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ELISA Protocol
3. Add sample. Incubate, Wash
Analyte = antibody
Analyte = antigen
4. Add conjugate. Incubate, Wash
E
E
Analyte = antibody
E
E
Analyte = antigen
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ELISA Protocol
6. Incubate, stop,
measure color change
5. Add substrate
ENZYME
Colourless
OD
Concentration
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Types of Enzyme Immunoassay
 Three main types:
1. Competitive ELISA
2. Indirect ELISA
3. Sandwich ELISA
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1- Competitive ELISA
• Enzyme activity is inversely proportional to the
concentration of the test substance
-9
• A sensitivity of nanograms (10 g)/ml can be
achieved
• This method can be used for measurement of
small molecules that are relatively pure
• such as insulin, and estrogen
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1- Competitive ELISA
Competition ELISA to detect Antigens (Antibody-coated plate)
Low [analyte]
E
E
E
1. Anti-analyte
Low [analyte]
Low [analyte]
High [analyte]
2. Analyte-E
+ sample
1. Anti-analyte
High [analyte]
E
3. Wash
2. Analyte- E
E + sample
1. Anti-analyte
4. Substrate
3. Wash
2. Analyte-E
+ sample
1. Analyte
E
E
High [analyte]
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2- Indirect ELISA
• Indirect ELISA is more sensitive than the
competitive
• Much assays are capable of detecting
concentrations of less than 1 pg/ml (10-12 g)/ml.
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2- Indirect ELISA
•
•
Screening of hybridoma supernatants
Detecting clinically important antibodies (eg. Autoantibodies)
E
E
E
E
E
E
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
1. Antigen
E
E
4. Substrate
3. Anti-(human) Ig-enzyme
2. Sample (human)
antibody
2. Sample (human) antibody
1. Antigen
1. Antigen
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3- Sandwich ELISA
• If the antibody is bound to the solid phase, these
assays are called sandwich immunoassays
• Antigens captured in these assays must have
multiple epitopes
• Used for detection of hormones, drugs, tumor
antigens, cytokines
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3- Sandwich ELISA
E
E
1. Anti-analyte
3. Anti-analyte-enzyme
2. Sample
1. Anti-analyte
2. Sample
1. Anti-analyte
4. Substrate
3. Anti-analyte-enzyme
2. Sample
1. Anti-analyte
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Lateral Flow Immunoassay
(LFIA)
(Immunochromatography System Assays)
(Membrane based immunodiagnostic assay)
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Principle
• Are based on the migration of nano or micro
particles on strips for analytes detection in
several areas
• In principle, any coloured particle can be used,
however latex (blue colour) or nanometer sized
particles of gold (red colour) are most commonly
used
• The technology is based on a series of capillary
beds, such as pieces of porous paper
• These elements has the capacity to transport fluid
(e.g., urine, plasma,..) spontaneously
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Principle
Immoblized Ab
(Control Line)
Monoclonal Ab
labelled with
colored
particles
Immoblized Ab
(Test Line)
support
Sample pad
• While the sample fluid start moving it dissolves particles and in one
combined transport action the sample and conjugate mix while
flowing through the porous structure
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Principle
Apply sample solution, upon application
of sample biochemicals dissolve
Negative: no antigen
Immobilised
Antibody area
Control area
Positive: antigen present
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• Uses
• Human and animal diagnostics Point of Care testing (PoCT)
• Forensics
• Environmental testing
• Advantages
• Rapid test (<15 minutes)
• Reliable and easy-to-use (no special equipment required nor
trained staff)
• Semi-quantitative or quantitative results
• Non-refrigerated storage
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Detection of HBsAg
Lateral Flow Immunoassay (LFIA)
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Intended Use
• HBsAg One Step Hepatitis B Surface Antigen
Test cassette (Serum/Plasma) is a rapid
chromatographic immunoassay for the qualitative
detection of Hepatitis B Surface Antigen in serum
or plasma
Summary and explanation
• Viral hepatitis is a systemic disease
primarily involving the liver, and in most
cases is caused by one of three viruses:
• Hepatitis A (HAV),
• Hepatitis B (HBV)
• or Hepatitis C (HCV).
• The antigen found on the envelope of HBV
is designated Hepatitis B Surface antigen
(HBsAg) and its presence in serum or
plasma indicates active HBV infection.
• In a typical Hepatitis B infection, HBsAg will
be detected 2 – 4 weeks before ALT levels
become abnormal and 3-5 weeks before
symptoms or jaundice develop.
Principle
• The membrane is precoated with anti-HBsAg
antibodies on the test line region of the test
• During testing, the serum or plasma specimen reacts
with the particle coated with anti-HBsAg antibody
• The mixture migrates upward on the membrane
chromatographically by capillary action to react with
anti-HBsAg antibodies on the membrane and
generate a colored line
• The presence of this colored line in the test region
indicates a positive result, while its absence indicates
a negative result
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Quality Control
• A procedural control is included in the test
• A red line appearing in the control region (C) is
the internal procedural control
• It confirms sufficient specimen volume and
correct procedural technique (membrane wicking
has occurred)
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Limitations
• LINEAR HBsAg cannot detect less than 1 ng/mL of
HBsAg in specimens
• If the test result is negative and clinical symptoms
persist, additional follow-up testing using other
clinical methods is suggested
• A negative result at any time does not preclude the
possibility of Hepatitis B infection
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Procedure
Allow the test strip to equilibrate to room temperature
prior to testing
2. Place the test device on a clean and level surface. Add
100 µl of serum or plasma to the specimen well (S) and
then start the timer.
3. Wait for the red line (s). The result should be read at 15
minutes
1.
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Interpretation
Control line
Test line
Positive
Negative
Invalid
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