Labeled Immunoassays
Part 2
Enzyme Linked Immunosorbent
Assay (ELISA)
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.
Enzyme Linked Immunosorbent Assay
(ELISA)
• One of many assays collectively called enzyme
immunoassays (EIA).
• The enzyme used an indicator molecule.
• The enzyme does not provide detection directly.
• The action of the enzyme on a substrate that
results in a change in the properties of the
solution.
• The substrate is brokendown by the enzyme to
produce chromogenic, fluorogenic or
luminescent products.
ELISA
• Can Be Used To Detect Both Antibody and Antigen
• Very Sensitive, ng & pg/mL
• Relies on Monoclonal Abs
Enzymes used in ELISA
• Enzymes used as labels for immunoassay are
chosen according to the following criteria:
 number of substrate molecules converted per
molecule of enzyme
 purity
 sensitivity
 ease and speed of detection
 stability
 absence of interfering factors in patient samples
 in addition, availability and cost of enzyme and
substrate.
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
chromogenic substrate to coloured product) resulting in
high sensitivity.
• An array of chromogenic, fluorogenic and
chemiluminescent substrates is available for use with
either enzyme.
• 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.
Enzyme Substrates
• A large selection of substrates is available for
performing the ELISA with an HRP or AP
conjugate.
• A common ELISA substrate for HRP:
– tetramethylbenzidine (TMB),
– TMB yields a blue reaction product that is watersoluble and absorbs light at 450 nm.
• The most common ELISA substrate for alkaline
phosphatase:
– the chromogen p-nitrophenyl phosphate (PNPP)
– PNPP yields a yellow reaction product that is watersoluble and absorbs light at 405 nm.
Linkage of Enzyme
• The enzyme label is linked to antibody or
analyte by several means.
 Glutaraldehyde is often used as a crosslinker
to join amino groups of the enzyme and the
molecule to be labeled.
 Maleimide derivatives are also used to attach
the enzyme label.
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.
• 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
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.
 Block unoccupied sites with a blocking agent
(200-300 μl/well) Store plate at 4°C with a
desiccant for future use.
ELISA Protocol
1- Coat solid phase with
•antigen when analysing antibody
•antibody when analysing antigen
Analyte = antibody
Analyte = antigen
Incubate, wash
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
 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.
 An individual blocking buffer will not be compatible with
every system; a variety of blockers in both Tris buffered
saline (TBS) and phosphate buffered saline (PBS) are
available.
 The proper choice of blocker for a given assay depends
on:
 the antigen itself
 and on the type of enzyme conjugate to be used.
(eg. AP + Blocking Buffer in TBS, PBS interferes with AP)
ELISA Protocol
2- Block free binding sites. Incubate. Wash.
Analyte = antibody
Analyte = antigen
ELISA Protocol
3. Add sample. Incubate. Wash
Analyte = antibody
Analyte = antigen
ELISA Protocol
4. Add conjugate. Incubate. Wash.
E
E
Analyte = antibody
E
E
Analyte = antigen
5. Add substrate
6. Incubate, stop, measure colour change
ENZYME
Colourless
OD
CONCENTRATION
Types of Enzyme Immunoassay
A- Heterogenous Enzyme Immunoassay
 Three main methods:
1. Competitive ELISA
2. Indirect ELISA
3. Sandwich ELISA
1- Competitive ELISA
• Enzyme activity is inversely proportional to
the concentration of the test substance.
• A sensitivity of nanograms (10-9 g)/ml can
be achieved.
• This method can be used for
measurement of small molecules that are
relatively pure,
• such as insulin
• and estrogen.
1- Competitive ELISA
COMPETITION ELISA TO DETECT ANTIGENS
(antibody-coated plate)
1. Anti-analyte
1- Competitive ELISA
Low [analyte]
High [analyte]
2. Analyte-E
+ sample
1. Anti-analyte
1- Competitive ELISA
Low [analyte]
High [analyte]
3. Wash
E
E
E
2. Analyte-E
E E
+ sample
1. Anti-analyte
E
E
1- Competitive ELISA
Low [analyte]
High [analyte]
4. Substrate
3. Wash
2. Analyte-E
+ sample
1. Analyte
2- Noncompetitive ELISA or
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.
2- Indirect ELISA
•
•
screening hybridoma supernatants
detecting clinically important antibodies
- autoantibodies
- anti-pathogens
- anti-allergens
1. Antigen
2- Indirect ELISA
2. Sample (human) antibody
1. Antigen
2- Indirect ELISA
E
E
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
E
E
E
E
2- Indirect ELISA
4. Substrate
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
E
E
E
3- Sandwich ELISA
• If the antibody is bound to the solid phase,
• These assays are called sandwich
immunoassays or capture assays
• Antigens captured in these assays must
have multiple epitopes.
3- Sandwich ELISA
eg. hormones
drugs
tumour antigens
cytokines
1. Anti-analyte
3- Sandwich ELISA
2. Sample
1. Anti-analyte
3- Sandwich ELISA
E
3. Anti-analyte-enzyme
2. Sample
1. Anti-analyte
E
3- Sandwich ELISA
3. Or:
anti-analyte-biotin
followed by
streptavidin-enzyme
2. Sample
1. Anti-analyte
E
S
E-S B S-E
E
S
E-S B S-E
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
4. Substrate
3. Or:
anti-analyte-biotin
followed by
streptavidin-enzyme
2. Sample
1. Anti-analyte
Membrane based cassette
assays
• This type of ELISA is
 rapid,
 easy to perform
 and give reproducible results.
• Designated primarily for primary care clinics and
home testing
• Many of these have been modified for increased
sensitivity and can be made semiquantitative for
use in a clinical laboratory by reading the color
reaction with a densitometer.
Membrane based cassette assays(Dipsticks)
Apply sample solution, upon application
of sample biochemicals dissolve
Positive: no antigen
Immobilised
Antibody area
Control area
Negative: antigen present
• Immunochromatography
• Biochemical components are separated across an absorbent
membrane into discrete distinct regions.
QUALITATIVE TEST
Control
Monoclonal Ab labeled
with enzyme
Immoblized Ab
+ substrate
support
Sample pad
B- Homogenous Enzyme Immunoassay
• 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 easier and faster to perform.
• Homogenous methods are generally less sensitive than
heterogeneous assays.
• They are based on the principle of change in enzyme
activity as specific antigen-antibody combination occurs.
• Enzymatic activity can be altered by steric exclusion of
the substrate, or there may be changes in the
conformation structure of the enzyme.
• Enzymes of this type include lysozyme, amaylase,
trypsin and others.
1- Enzyme-multiplied immunoassay
• It is based on the principle of change in enzyme activity
as specific antigen-antibody combination occurs.
Reagent antigen is labeled with an enzyme tag. When
antibody binds to specific determinant sites on the
antigen, the active site on the enzyme is blocked,
resulting in a measurable loss of activity.
• Free analyte (antigen) competes with enzyme-labeled
analyte for a limited number of antibody-binding sites, so
this is a competitive assay. Enzyme activity is directly in
proportion to the concentration of patient antigen or
hapten present in the test solution. A physical separation
of bound and free analyte is thus not necessary.
Advantages & Disadvantages
• Advantages of ELISA




High sensitivity
No need for expensive instruments
Reagents are inexpensive and have a long shelf time
No disposal problems, or health hazard of radiation
• Disadvantages of ELISA
 Some specimens may contain natural inhibitors
 Size of enzyme may be a limiting factor in the design
of some assays
 Nonspecific binding and sensitivity of enzymes to
temperature
Toxoplasma gondii
• Toxoplasma gondii is a small intracellular parasite,
whose life cycle has a sexual and an asexual phase.
• Sexual development is restricted to the intestinal cells of
(probably exclusively) cats; the oocysts formed are
excreted and due to their resistant cell walls they may be
infectious under advantageous circumstances for at least
1 year.
• Animals and man are intermediate hosts for the asexual
proliferation of T. gondii: the ingested parasites will
proliferate explosively within the host cells lysing them
eventually.
• They disseminate throughout the body via circulation
and lymphatic system and may infect any cell type.
Cysts are virtually impossible to be killed in the
intermediate host.
Toxoplasma gondii
• Most T. gondii infections are asymptomatic or present
with a transient
• febrile illness, often with lymphadenopathy.
• Primary infections acquired during pregnancy can cause
•
•
•
•
•
stillbirth or congenital abnormalities,
including blindness,
encephalomyelitis,
impaired brain functions
and convulsions in the newborn.
• Infections can also be more severe in individuals who
are infected with HIV or who are otherwise
immunocompromised.
• Toxoplasmosis can be treated with antibiotics but early
detection of the disease is crucial for effective therapy.
Test Principle
• Microtiter wells are coated with antigens.
• This constitutes the solid phase.
• Patient's sample is added to the wells and any
antibodies specific for the antigen present will bind to the
solid phase.
• After removal of unbound material, anti-human IgG or
IgM conjugated to an enzyme (alkaline phosphatase)
is allowed to react with the immune complex.
• After removal of excess conjugate by washing, an
appropriate substrate (paranitrophenylphosphate) is
added, with which the conjugated enzyme reacts
producing a coloured derivative of the substrate.
• The colour intensity is proportional to the level of specific
antibody bound and can be quantified photometrically.
Test procedure
• Place the required number of cavities in the frame and
prepare a protocol sheet.
• Prepare the diluted washing solution
• Wash microplate strips once with working washing solution.
Remove remaining liquid by tapping on absorbent paper
• Dilute test sera by adding 10 µl of sample to 1 ml of diluent
• Calibrators 0, 6, 60 and 240 IU/ml are diluted in the same
manner
• Add each 200 μl of diluted sample or calibrators into the
appropriate wells
– well A1 0 IU/ml
– well B1 6 IU/ml
– well C1 60 IU/ml
– well D1 240 IU/ml
– well E1, F1, G1, etc.. 200 µl of diluted sample
Test procedure
•
•
•
Sample incubation for 60 minutes (+/- 5 min) at
37°C (+/- 1°C).
Prepare working conjugate 15 minutes before
the end of first incubation period
After incubation wash all wells with washing
solution (by automated washer or manually):
•
•
•
•
•
aspirate or shake out the incubation solution
fill each well with 300 μl washing solution
aspirate or shake out the washing buffer
repeat the washing procedure 2 times (altogether 3 times!)
dry by tapping the microtest plate on a paper towel
Test procedure
•
Addition of conjugate
•
•
•
•
Add 200 μl of working-conjugate to the
appropriate well
Conjugate incubation for 60 minutes (+/- 1 min) *
at 37°C (+/- 1°C)
After incubation wash all wells 4 times with
washing solution (see above)
Addition of substrate
•
•
Add 200 μl substrate solution to each well
Substrate incubation for 30 minutes (+/- 1 min) *
at 37°C (+/- 1°C) in the dark
Test procedure
•
Stopping of the reaction
•
•
Add 100 μl stopping solution to each well, shake
microtest plate gently to mix.
Read optical density
•
Read OD within 30 minutes at 450 nm against
substrate blank
Results
• The presence and quantity of IgG antibodies to
T. gondii in the test sample is determined by
comparing the optical density (OD) of the test
sample to a standard range.
• For validation of the test the following criteria
must be met:
• OD values
– OD R3 ≤ 0.200
– OD R4c ≥ 1.000
• Ratio:
– OD R4a / OD R3 ≥ 2
Results
• After drawing the curve from the concentration of
standards on the x-axis with absorbance of
standards on the y-axis, find the value
corresponding to the OD reading of the test
sample on the y-axis and draw a horizontal line
to the standard curve. At the point of intersection
with the standard curve draw a vertical line to
the x-axis. Read the concentration in IU/ml at the
point of intersection with the x-axis.
• Interpretation of results
• Negative result: titer < 6 IU/ml
• Positive result: > 9 IU/ml