Enzyme Linked Immunosorbent Assay

(ELISA)
Introduction
• Quantitative Immunological procedure
• Discovered by Engrall & Perlma in 1971
• Estimating ng/ml to pg/ml.
Definitions
Antigens
A substance that when introduced into the body
stimulates the production of an antibody

Immunoassay
A laboratory technique that makes use of the
binding between an antigen and its homologous
antibody in order to identify and quantify the
specific antigen or antibody in a sample
• Enzyme Conjugate

Enzyme attached irreversibily to Antibody

Definitions
• Antibodies (also known as immunoglobulins
abbreviated Ig) are gamma globulin proteins that
are found in blood and are used by the immune
system to identify and neutralize foreign objects,
such as bacteria and viruses.
• Response to Antigenic Stimuli
History
The first methods were based on Agglutination methods.
Reaction between Antibody &Antigen Agglutination occurs
Characterised by gel formation in liquid phase or
an opaque band in an agar plate assay
Agglutination occurs only when there is right amounts of Antigen &
Antibody.
Antibody has two binding sites then each of them can be bound to
different antigen particles.
Zone of Equivalence
• The binding of antibody (Ab) to antigen (Ag) for the specific and
sensitive detection of an analyte.
• The Ag–Ab interactions may involve unlabeled reactants in less
analytically sensitive techniques or a labeled reactant in more
sensitive techniques.
• If too much antibody is present then each antigen molecule will bind
multiple antibodies and the meshwork will not develop.
• If too much antigen is present then each antibody will bind only one
antigen particle and no lattice will form.
• For this reason a dilution series of antibody is often made and a
measure of antigen concentration can be made from the end point at
which agglutination occurs.
• As this happens bridges are formed created by the antibody
molecules spanning two antigen molecules.
• The resulting lattice that is created forms a stable structure where
antigen and antibody particles are suspended in solution by their
attachment to each other.
• For this to take place there must be a precise amount of antigen and
antibody present and this is known as equivalence
• Outside the zone of equivalence, the amount of precipitate is
diminished or absent because the ratio of Ab to Ag is out of
proportion and the cross-linking of Ag is decreased.
• When Ab concentration is in excess and cross-linking is decreased,
the assay is in prozone. Conversely, when Ag concentration is in
excess and crosslinking is decreased, the assay is in postzone.
 ELISA

• Enzyme Linked Immunosorbent Assay (ELISA)

• Term Was Coined By Engvall and Pearlmann in 1971
• Different Types
• Sandwich
• Indirect
• Competitive
• Similar To RIA, Except No Radiolabel
• Can Be Used To Detect Both Antibody and Antigen
• Very Sensitive, pg/mL
• Relies on Monoclonal Abs
Types
• Non Competitive
• Direct
• Indirect
• Sandwich
• Competitive
• Multiple & Portable
Competitive ELISA
• The labelled antigen competes for primary
antibody binding sites with the sample antigen
(unlabeled).
• The more antigen in the sample, the less
labelled antigen is retained in the well and the
weaker the signal).
• In one of the simplest unlabeled immunoassays introduced into the
clinical laboratory,
• unlabeled Ab was layered on top of unlabeled Ag (both in the fluid ph
the Ab and Ag diffused and the presence of precipitation was
recorded); during the incubation period,
• the Ab and Ag diffused and the presence of precipitation was
recorded.
• When the Ag–Ab complex is of sufficient size, the interaction with
water is limited so that the complex becomes insoluble and
precipitates
• It has been observed that if the concentration of Ag is increased while
the concentration of Ab remains constant, the amount of precipitate
formed is related to the ratio of Ab to Ag.
• There is an optimal ratio of the concentration of Ab to the
concentration of Ag that results in the maximal precipitation; this is
the zone of equivalence.
• Gel is dilute agarose (typically less than 1%) dissolved in an aqueous
buffer. This provides a semisolid medium through which soluble Ag
and Ab can easily pass.
• Precipitated immune complexes are easier to discern in gel versus a
liquid suspension.
• using electrophoresis and are summarized in
• The simplest and least sensitive method is double diffusion (the
Ouchterlony technique).
• Agarose is placed on a solid surface and allowed to solidify. Wells are
cut into the agarose.
• A common template is six Ab wells, surrounding a single Ag well in the
center. Soluble Ag and soluble Ab are added to separate wells and
diffusion occurs.
Ouchterlony Double Diffusion
• The Ouchterlony double diffusion agar plate method is the
commonest gel-based assay system used .
• Wells are cut in an agar plate which is then used to load samples and
an antibody solution.
• The antigens and the antibody diffuse through the agar and if the
antibody recognises antigen within the gel then a precipitin band is
formed.
• A diffusion gradient is formed through the agar as the reagents
progress and so there is no requirement for dilutions to be made to
ensure that agglutination occurs.
• the precipitin band of an unknown sample is compared with the
precipitin band of a sample known to contain the Ab.
• A pattern of identity confirms the presence of the Ab in the unknown
sample.
• Patterns of partial identity and nonidentity are ambiguous. This
technique can be used to detect Abs associated with autoimmune
diseases, such as Sm and RNP detected in systemic lupus erythematosus,
SSA and SSB in Sjögren’s syndrome, and Scl-70 in progressive systemic
sclerosis.
• However, most of these Abs are also detected using enzyme-linked
immunoassays or multiplex bead array systems (Luminex).
• Polyclonal antibodies can be made to different subspecies of bacteria
and these will recognise surface epitopes on them.
• Because the subspecies are related then they will share some surface
epitopes and the closer they are related the more they will share.
• Antibodies made to one organism will therefore react to a greater or
lesser degree to a related organism depending on how many surface
epitopes they share.
Immunoassay
• By far, the vast majority of immunoassays carried out fall into the
category of enzyme immunosorbent assays.
• These are routinely used for the diagnosis of infectious agents
• such as viruses, and other substances in blood.
• radial immunodiffusion (RID), is an immune precipitation method
used to quantitate protein (the Ag).
RID
• In this method, monospecific antiserum is added to the liquefied
agarose;
• then the agarose is poured into a plate and cooled. Wells are cut into
the solidified agarose.
Multiple standards, one or more quality control samples, and patient
samples are added to the wells.
The Ag diffuses from the well in all directions, binds to the soluble Ab in
the agarose, and forms a complex seen as a concentric precipitin ring
RID
• The diameter of the ring is related to the concentration of the Ag that
diffused from the well. A standard curve is constructed to determine
the concentration in the quality control and patient samples.
• The usable analytic range is between the lowest and highest
standards.
• If the ring is greater than the highest standard, the sample should be
diluted and retested. If the ring is less than the lowest standard, the
sample should be run on a lowlevel plate.
RID
• Two variations exist:
• the endpoint (Mancini) method4 and

the kinetic (Fahey-McKelvey) method.5

The endpoint method requires that all Ag diffuse from the well and the concentration of the Ag is related
to the square of the diameter of the precipitin ring;
• the standard curve is plotted on linear graph paper and is the line of best fit.
• To ensure that all of the Ag has diffused, the incubation time is 48 to 72 hours, depending on the
molecular weight of the Ag; for example, IgG quantitation requires 48 hours, and IgM requires 72 hours.
• In contrast, the kinetic method requires that all rings be measured at a fixed time of 18 hours; a sample
with a greater concentration will diffuse at a faster rate and will be larger at a fixed time.
• Using semilogarithmic graph paper, the concentration of the Ag is plotted against the diameter of the
precipitin ring; the line is drawn point to point. For those performing RID, the endpoint method is
favored because of its stability and indifference to
RID
• For those performing RID, the endpoint method is favored because of
its stability and indifference to
• temperature variations; however, turnaround time is longer compared
with the kinetic method.
Counter Immuno Electrophoresis
• Counterimmunoelectrophoresis is an immune precipitation method
that uses an electrical field to cause the Ag and Ab to migrate toward
each other.
• Two parallel lines of wells are cut into agarose; Ab is placed in one line
and Ag is placed in the other. Ab will migrate to the cathode and the
Ag to the anode; a precipitin line forms where they meet.
• This qualitative test is useful to detect bacterial antigens in
cerebrospinal fluid and other fluids when a rapid laboratory response
is needed.
IEP & IFE
• Immunoelectrophoresis (IEP) and immunofixation electrophoresis (IFE) are two methods used
in the clinical laboratory to characterize monoclonal proteins in serum and urine. In 1964,
Grabar and Burtin6 published methods for examining serum proteins using electrophoresis
coupled with immunochemical reactions in agarose.
• Serum proteins are electrophoretically separated and then reagent Ab is placed in a trough
running parallel to the separated proteins.
• The Ab reagent and separated serum proteins diffuse; when the reagent Ab recognizes the
serum protein and the reaction is in the zone of equivalence, a precipitin arc is seen.
• The agarose plate is stained (typically, with a protein stain such as Amido black 10), destained,
and dried to enhance the readability of precipitin arcs, especially weak arcs.
• The size, shape, density, and location of the arcs aid in the interpretation of the protein. All
interpretation is made by comparing the arcs of the patient sample with the arcs of the quality
control sample, a normal human serum. Because IEP is used to evaluate a monoclonal
• related to the concentration of suspended particles and path length.
Nephelometers measure light at an angle other than 180 degrees from the
incident light; most measure forward light scattered at less than 90 degrees
because the sensitivity is increased.
• The relative concentration of the Ab reagent and Ag is critical to ensure that
the size of the complex generated is best detected by the nephelometer or
turbidimeter and that the immune reaction is not in postzone or prozone.
Therefore, it may be important to test more than one concentration of
patient sample, to monitor the presence of excess Ab, or to add additional
antiserum and monitor the peak rate. Excess Ab would indicate that there is
little Ag and that the reaction is underestimated.
•
• For both turbidimetry and nephelometry, all reagents and sera must be free of
particles that could scatter the light. Pretreatment of serum with polyethylene
glycol, a nonionic, hydrophilic polymer, enhances the Ag–Ab interaction. Because
the polymer is more hydrophilic than the Ag or Ab, water is attracted from the Ag
and Ab to the polyethylene glycol.
• This results in a faster rate and greater quantity of Ag–Ab complex formation. Both
methods can be performed in an endpoint or a kinetic mode. In the endpoint mode,
a measurement is taken at the beginning of the reaction (the background signal)
and one is taken at a set time later in the reaction (plateau or endpoint signal); the
concentration is determined using a calibration curve. In the kinetic mode, the rate
of complex formation is continuously monitored and the peak rate is determined.
The peak rate is directly related to the concentration of the Ag, although this is not
necessarily linear. Thus, a calibration
• Labeled Immunoassays General Considerations In all labeled immunoassays, a reagent (Ag or Ab) is usually
labeled by attaching a particle or molecule that will better detect lower concentrations of Ag–Ab complexes.
• Therefore, the label improves analytic sensitivity. All assays have a binding reagent, which can bind to the Ag or
ligand. If the binding reagent is an Ab, the assay is an immunoassay.

• If the binding agent is a receptor (e.g., estrogen or progesterone receptor), the assay is a receptor assay. If the
binding reagent is a transport protein (e.g., thyroxine-binding globulin or transcortin), the assay may be called
competitive protein-binding assay.
• are used today almost exclusively, with two notable exceptions: estrogen and progesterone receptor assays and
the thyroid hormone–binding ratio, which uses thyroxine-binding globulin. Immunoassays may be described
based on the label— which reactant is labeled,
• the relative concentration and source of the Ab, the method used to separate free from bound-labeled reagents,
the signal that is measured, and the method used to assign the concentration of analyte in the sample.
• Immunoassay design, therefore, has many variables to consider, leading to diverse assays. Labels The simplest
way to identify an assay is by the label used. Table 7-2 lists the commonly used labels and the methods used to
detect the label.
• In this technique the antigen is immobilised on to a solid phase, either the reaction
vessel or a bead.
• The most commonly used solid phase is the enzyme linked immunosorbent assay
(ELISA) plate. Immobilisation is achieved by the use of a coating antibody which
actively traps antigen to the solid phase.
• A second antibody (antibody enzyme conjugate) which is labelled with a reporter
enzyme is allowed to bind to the immobilised antigen.
• The enzyme substrate is then added to the antigen/ antibody/enzyme complex and
a reaction, usually involving a colour change, is seen. There are many permutations
of this method but all of them rely on the antibody–antigen complex being formed
and the presence of it being confirmed by the reactions of the reporter enzyme.
•
• These assays rely on a stepwise addition of layers with each one being linked
to the one before.
• The antigen is central to the assay as it provides the bridge between the
solid phase and the signalgenerating molecule.
• Without antigen, the antibody enzyme conjugate cannot be bound to the
solid phase and no signal can be generated.
• The coating antibody also concentrates the antigen from the sample as it
binds the antigen irreversibly and so the coating layer has the ability to
concentrate available antigen until saturation has been reached.
• This is particularly useful when testing for low levels of antigens in fluids
such as blood serum.
DAS ELISA
• Double antibody sandwich (DAS) ELISA is probably the most widely
used immunochemical technique in diagnostics
• It is rapid, robust, and reliable and can be performed and the results
interpreted with minimal training.
Principle
• The antigen is immobilised to a solid phase by a primary antibody
and detected with a second antibody which has been labelled with a
marker enzyme.
• The antigen creates a bridge between the two antibodies and the
• presence of the enzyme causes a colour change in the chromogenic
(colour-producing) substrate.
• The marker enzyme used is usually either horseradish peroxidase
(HRP) or alkaline phosphatase (AP).
IRMA
• Insome systems the enzyme is replaced with a radioactive label and
this format is known as the immunoradiometric assay (IRMA
Applications
• DAS ELISA is used extensively in horticulture and agriculture .
• Potato tubers that are to be used as seed for growing new crops have
to be free of potato viruses and
• screening for this is carried out by DAS ELISA.
• There are many potato viruses but potato leafroll virus (PLRV) in
particular causes considerable problems.
• PLRV antibodies are coated onto the wells of ELISA plates and then
the sap to be tested is added.
• After incubation, the plates are washed and PLRV antibody
conjugated to alkaline phosphatase is added.
• The plates are incubated and after washing, substrate is used to
identify the positive wells.
• The system again requires the presence of the antigen (PLRV) for the
sandwich of antibodies to be built up.
TAS
Triple Antibody Sandwich test
• Triple antibody sandwich (TAS) ELISA, also known as indirect ELISA, is
a widely used method
• It is often used to identify antibodies in patient blood which may be
there as the result of infection.
• As with other immunoassays, layers of reagents are built up, each
dependent on the binding of the previous one
• The system is used to test patient blood for the presence of hepatitis
B virus (HBV) antibodies as a diagnostic test for this disease.
• The system is used to test patient blood for the presence of hepatitis
B virus (HBV) antibodies
• as a diagnostic test for this disease
• In this test HBV coating antibody is bound to the wells of a microtitre
plate and HBV coat protein added to them.
• The live virus is not used as antigen as this would be too dangerous to
use in the laboratory.
HBV coat protein is made synthetically specifically for use as antigen
in this type of test.
After incubation and washing, patient serum is added which if it
contains antibodies reacts to the antigen.
• Anti-human antibody conjugated to an enzyme marker is then added
which will bind to the patient antibodies.
• Substrate is then added to identify samples which were positive.
• The test works well for the diagnosis of HBV infection and is also used
to ensure that blood donations given for transfusion are free from this
virus.
Enhanced assays
• The maximum sensitivity of ELISA is in the picomole range.
• The physical limitations are based on the dynamics of the double
binding event and
• the subsequent generation of signal above the background substrate
value.
• Most workers have concentrated their efforts on the amplification of
signal.
• Antibody binding cannot be improved as it is primarily a random
event modified by the individual avidities of the antibodies
themselves.
• temperature modification
• There are two basic ways that signal can be amplified in ELISA.
• More enzyme can be bound by using multivalent attachment
molecules.
• Systems using the avidin–biotin binding system allow amplification
through this route.
• Both avidin and biotin are tetravalent (i.e. they have four binding
sites) and it is this property that produces the amplification.
• Both avidin and biotin are tetravalent (i.e. they have four binding
sites) and it is this property that produces the amplification.
• The detection antibody is labelled with biotin and the reporter
enzyme with avidin.
• The high affinity and multivalency of the reagents allows larger
complexes of enzyme to be linked to the detection antibody,
producing an increase in substrate conversion and improved colour
development in positive samples.
• The alternative amplification step is by enhancing the substrate
reaction usually by using a double enzyme system.
• Both avidin and biotin are tetravalent (i.e. they have four binding
sites) and it is this property that produces the amplification.
• The detection antibody is labelled with biotin and the reporter
enzyme with avidin.
• The high affinity and multivalency of the reagents allows larger
complexes of enzyme to be linked to the detection antibody,
producing an increase in substrate conversion and improved colour
development in positive samples
• The primary enzyme bound to the antigen catalyses a change in the second
enzyme which then generates signal.
• Both of these methods will increase the signal generated but may also increase
the background reaction.
• Alkaline phosphatase conjugated secondary enzyme can be used to drive a
secondary reaction involving NADP dephosphorylation to NAD which is further
reduced to NADH by alcohol dehydrogenase.
• This in turn creates a loop in which a tetrazolium salt is oxidised as the NADH
returns to NAD.
• The tetrazolium salt is chromogenic when in the oxidised state. The cyclic nature
of the reaction causes the amplification and increases the observed colour
development
• Some claims have been made for ‘supersubstrates’ which work
directly with enzyme–antibody conjugates but there is usually little in
the way of true gain if standard curves are calculated for the various
substrate types
• Competeitive Immunoassay
• It is quantitative when used in conjunction with a standard curve.

The principle is based on competition between the natural antigen (hormone) to be

tested for and an enzymeconjugated form of the antigen which is the detection
reagent.
The test sample and a defined amount of enzyme-conjugated antigen are mixed
together and placed into the coated wells of a microtitre plate. T
he antigen and conjugated form of it compete for the available spaces on the coating
antibody layer. The more natural antigen present the more it will displace (compete
out) the conjugated form leading to a reduction in enzyme bound to the plate.
The relationship of substrate colour development is therefore inverted; the more
natural antigen bound the lower the signal generated.
This form of ELISA is routinely used for testing blood samples for thyroxin.
Thyroxin is a hormone that is responsible for regulating metabolic rate and
deficiencies (hypothyroidism) and excesses (hyperthyroidism) of it will
slow or speed up the metabolism.
Patients can be given additional thyroxin if required if they are deficient
but it is important to establish the baseline level before treating the
condition.
Competitive ELISA is used for this as an accurate measure of the
circulating level of the hormone can be made from a standard curve of
known dilutions.
• In some assays the enzyme is replaced with a radioactive label
• and this form of competitive ELISA is known as the radioimmunoassay
(RIA).
• Competitive ELISA using conjugated antibody can also be used to
quantify levels of circulating antibody in test serum.
• The solid phase has the antigen to which the antibody will attach
directly coated on to it.
• The test serum and the conjugated form of antibody are mixed
together and added to the reaction wells.
• The conjugated and test antibody then compete to bind to the
antigen.
• The level of antibody can again be determined by the reduction in
signal observed by addition of the substrate.
• The assay is concluded by adding an enhancement solution which
causes dissociation of the lanthanide from the antibody molecules.
• The signal is generated by stimulating the lanthanide with light of a
specific wavelength and measuring the
The Name
Instrumentation
• How many wells in ELISA
TYPES
• Enzyme linked Immunoassay
• Enzyme multiplied Immunoassay
• Cloned Enzyme donor Immuno Assay
• Fluro Immuno Enzymatic Methods.
Principles
Operation
Labels in ELISA
• Horseradish Peroxidase
• Alkaline Phosphatase
• Glucose 6Dehysrogenase
• βGalactosidase
Sample Requirement
Maintenance
Calibration
RLUs- Relative Light Units
Advantages of RadioImmunoassay
Advantages of CLIA
DEFLIA
• DELFIA is a time-resolved fluorometric assay which relies on the
unique properties of lanthanide chelate antibody labels
• The lanthanides will generate a fluorescent signal when stimulated
with light of a specific wavelength
• DELFIA offers a signal enhancement greater than that possible from
conventional enzyme-linked assays.
• The lanthanide chelates are conjugated onto the secondary antibody
and as there are a number of lanthanides each with a unique signal
which can be used, multiplexing (more than one test carried out in
the same reaction vessel) is possible.