ENZYME IMMUNO ASSAY
S Rubesh Kumar M.Pharm, (Ph.D),
Department of Pharmaceutical Analysis
J N T U A – O T R I
ANANTAPUR - 515001
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ENZYME IMMUNO ASSAY
EIA: Assay systems involving use of antigens, haptens, or antibodies labeled with
an enzyme have recently been applied to the measurement of substances in
biological fluids. These assay systems have been given various names: enzyme-,
enzymic-, enzymatic-, and enzymoimmunoassay (EIA),’ enzyme-linked immunoassay,
enzyme-labeled immunoassay, enzyme-coupled immunoassay, immunoenzymatic
assay, and enzymelinked immunosorbent assay (ELISA). In addition, there is a type
of EIA, Homogeneous EIA, in which, unlike the other types, separation of free and
bound labeled material is not required. Homogeneous EIA has also been called the
enzyme multiplied immunoassay technique (EMIT) and enzyme-inhibition
immunoassay.
Immunoassays combine the principles of chemistry and immunology enabling
scientific tests, e.g. enzyme immunoassays and immunoblotting for a specific and
sensitive detection of the analytes of interest. The basic principle of these assays
is the specificity of the antibody-antigen reaction. Though being very specific and
sensitive immunoassays are easy to perform which has contributed to the
widespread use and tremendous success. In immune blotting like the Western Blot
of electrophoretically separated proteins a single one can be identified by its
antibody.
RIAs (Radioimmunoassay) and enzyme immunoassays like ELISA (Enzyme-linked
immunosorbent assay), LIA (luminescent immunoassay), and FIA (fluorescent
immunoassay) are widely used in research, drug discovery and diagnostics for
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highly specific and cost efficient detection of analytes not detectable with other
techniques.
The antibodies – either used as primary or secondary ones – are labelled with
radioisotopes (e.g. 125I), fluorescent dyes (e.g. FITC) or enzymes (e.g. HRP or AP)
which catalyse fluorogenic or luminogenic reactions.
Immunoassays involve tests using antibodies as reagents.
Enzyme immunoassays (EIA) make use of enzymes attached to one of the
reactants in an immunoassay to allow quantification through the development of
color after addition of a suitable substrate/chromogen.
As indicated above ELISAs involve the step-wise addition and reaction of reagents
to a solid phase bound substance, through incubation, and separation of bound and
free reagents using washing steps.
An enzymic reaction is utilized to yield color and is used to quantify the reaction,
through use of an enzyme labeled reactant.
A very brief definition of terms used in ELISA is needed.
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.
i) The Antibody: An immunoglobulin, a specialized immune protein, produced
because of the introduction of an antigen into the body, and which
possesses the remarkable ability to combine with the very antigen that
triggered its production (specific affinity)
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ii) The antibody recognises and bind to the antigenic determinant region of
the antigen
Antigens A substance that when introduced into the body
stimulates the production of an antibody. An antigen is any substance that
causes the immune system to produce antibodies against it. The substance
may be from the environment or formed within the body. The immune system
will kill or neutralize any antigen that is recognized as a foreign and
potentially harmful invader. The term originally came from antibody
generator ]and was a molecule that binds specifically to an antibody, but the
term now also refers to any molecule or molecular fragment that can be
bound by a major histocompatibility complex (MHC) and presented to a T-
cell receptor. "Self" antigens are usually tolerated by the immune system;
whereas "Non-self" antigens can be identified as invaders and can be
attacked by the immune system.
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
Analyte The sample being analyzed and in immunoasssays the analyte is
either Antibody or Antigen
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Types of Enzyme Immuno Assay
1. Competitive EIA for antigen: Labeled antigen competes with unlabeled antigen
for binding to a limited quantity of antibody. The antibody-bound antigen is
separated from the free antigen by the use of solid phase antibody or a second
antibody with specificity for the first. The enzyme activity in either the bound or
free fraction is determined and related to the concentration of the unlabeled
antigen. This procedure is analogous to the classical RIA method. In the sequential
saturation variant of the competitive assay, the addition of the labeled antigen is
delayed until the binding between the antibody and the unlabeled antigen is
complete. This method is analogous to the sequential RIA.
2. Immunoenzymometric: assay for antigen. Antigen reacts with excess labeled
antibody and, after incubation; excess solid-phase antigen is added. The solid-
phase antigen reacts with the free labeled antibody remaining and, after
separation of the solid-phase, the enzyme activity associated with soluble antigen
is measured and related to the concentration of antigen. This assay is analogous to
the immunoradiometric assay.
3. Sandwich EJA for antigen: This procedure requires the antigen to have at
least two binding sites. Antigen reacts with excess solid-phase antibody, and after
incubation followed by washing, the bound antigen is treated with excess labeled
antibody. After further washing the bound label is assayed, and this provides a
direct measure of the amount of antigen present. This assay is analogous to the
“two-site” immunoradiometric procedure. A variation of this method
(“double sandwich” EIA) involves a third antibody. This antibody carries the label
and reacts with unlabeled second antibody already bound to the antigen. As before,
the amount of antigen is found by measuring the amount of bound label.
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4. EIA for antibody. Antibody binds to excess solid-phase antigen and, after
incubation followed by washing, labeled second antibody with specificity for the
first antibody is added. The bound label is assayed after further washing and it
provides a direct measurement of the amount of specific antibody present.. This
assay is analogous to the radioallergosorbent technique. The system may also be
used to assay antigens.
5. Homogeneous EIA for haptens. This type of EIA does not require separation
of free and bound label, because the assay depends on inhibition or activation of
the enzyme label by antibody binding. In the first of these assays the haptenic
group, a morphine derivative, was attached to lysozyme. When antibodies to
morphine were bound to this modified lysozyme the enzyme was inhibited, possibly
because the large substrate, the cell wall peptidoglycan of Micrococcus luteus, was
unable to gain access to the catalytic site. However, when free morphine was
present it competed with the enzyme-bound morphine for the antibody binding
sites. Thus fewer antibodies were bound to the enzyme and consequently inhibition
of its catalytic activity was decreased. Work has also been done on an assay in
which a morphine derivative was attached to malate dehydrogenase. In this case
the inhibition of the enzyme’s action on binding to antibody appears to be caused
by a conformational change induced in the enzyme rather than by steric hindrance.
In a similar system, attachment of a thyroxine derivative to malate dehydrogenase
caused inhibition of the enzyme but this inhibition was reversed when antibodies
against thyroxine were bound to the modified enzyme.3 In this assay the presence
of free thyroxine prevents binding of the antibody to the enzyme and thus the
enzymic activity decreases.
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6. Other EIAs. Other types of EIA are possible and two additional EIAs for
antibodies have been briefly described . In the first (Type 6a), labeled and
unlabeled antibody compete for solid-phase antigen. In the second assay (Type 6b)
labeled antigen reacts with antibody; solid-phase antibody is then added and the
remaining labeled antigen reacts with it. The amount of antibody is determined in
both cases by measuring the enzyme activity in the free or bound fractions after
centrifugation.
ELISA technique
Is a biochemical technique used mainly in immunology to detect the presence of an
antibody or an antigen in a sample.
The technique is divided into
Direct ELISA Indirect ELISA
SandwichELISA Competitive ELISA
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Direct method
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Coating (Steps 2 and 3)
Antigen is diluted in a buffer and added to the solid phase, commonly high pH (9.6)
carbonate/bicarbonate buffer or neutral phosphate buffered saline. The key is
that the buffer contains no other proteins that might compete with the target
antigen for attachment to the plastic solid phase. Antigens are mainly protein in
nature, and attach passively to the plastic during a period of incubation. The
temperature and time of the incubation is not so critical, but standardization of
conditions is vital and the use of incubators at 370C is favoured, (since they are
widely available in laboratories).
Washing (Steps 4 and 7)
Washing is necessary to separate free from bound reagents. In Step 4, any
unadsorbed antigen is removed, in Step 7 it is necessary to remove free antibody
enzyme from that bound specifically to the antigen. Washing can be simply flooding
and emptying wells, using a neutral buffered solution (e.g., PBS).
Conjugate addition (Step 6)
Antibodies conjugated with an enzyme are added directed specifically against
antigenic sites on the solid-phase bound reagent. The conjugated antibodies are
diluted in a buffer containing some substances which inhibits passive adsorption of
protein, but which still allows immunological binding. Such substances are either
other proteins, which are added at high concentration to 'compete' for the solid
phase sites with the antibody protein; or detergents at low concentration and are
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termed "blocking " agents, and the buffers they help formulate are called "blocking
buffers". On incubation, antibodies bind to the antigen.
A simple washing step is then used to get rid of unbound antibodies (Step 7). The
complex then on plate is antigen combined with specific enzyme labeled antibody.
Substrate/Chromphore Addition (Step 9)
This involves addition of a suitable substrate or substrate/chromogen combination
for the particular enzyme attached to the antibodies. The objective is to allow
development of a color reaction through enzymic catalysis. The reaction is allowed
to progress for a defined period after which the reaction is stopped by the
alteration in pH of the system, or addition of an inhibiting reactant. Finally, the
color is quantified by the use of a spectrophotometer reading at the appropriate
wavelength for the color produced.
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Indirect ELISA
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Steps 1-5 are similar to the Direct system where wells are coated with antigen.
Step 6 involves the addition of unlabeled antibodies, which are diluted in a buffer to
prevent non-specific attachment of proteins in antiserum to solid phase, (blocking buffer).
This is followed by incubation and washing away excess (unbound) antibodies, to achieve
specific binding
Step 7 involves washing away unbound antibodies leaving the complex in Step 8, consisting
of antigen and antibody, attached to solid phase.
Step 9 is the addition of the conjugate (enzyme-labeled), anti-species antibodies, diluted
in blocking buffer, again followed by incubation and washing to achieve binding of
conjugate. After washing (Step 10) the complex on the solid phase has increased with the
enzyme conjugate detecting the specific species of antibody detecting the antigen (Step
11)
Step 12 involves the addition of the substrate/chromophore and allowing the colour to
develop (Step 13). Stopping the color is Step 14 and reading is Step 15.
The Indirect system is similar to the Direct system, in that antigen is directly attached to
the solid phase and 'targeted' by added antibodies, (detecting antibodies). However, these
added antibodies are not labeled with enzyme but are themselves 'targeted' by antibodies
linked to enzyme. Such antibodies are produced against the immunoglobulins of the species
in which the detecting antibodies are produced and termed "anti-species" conjugates.
Thus, if the detecting antibodies are produced in rabbits, the enzyme labelled antibodies
would have to be anti-rabbit immunoglobulin in nature. This allows great flexibility in use of
anti-species conjugates in that different specificities of conjugate can be used to detect
particular immunoglobulins binding in the assay and there are literally thousands of
commercially available conjugates available. For example, the anti-species conjugate could
be anti-IgM, anti IgG1, IgG2 , etc.
The Indirect system offers the advantage that any number of antisera can be examined
for binding to a given antigen using a single anti-species conjugate. Such systems have
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been heavily exploited in diagnostic applications particularly when examining (screening)
large numbers of samples.
The protein antigen to be tested for is added to each well of ELISA plate,
where it is given time to adhere to the plastic through charge interactions
A solution of non-reacting protein is added to block any plastic surface in
the well that remains uncoated by the protein antigen
Then the serum is added, which contains a mixture of the serum antibodies,
of unknown concentration, some of which may bind specifically to the test
antigen that is coating the well.
Afterwards, a secondary antibody is added, which will bind to the antibody
bound to the test antigen in the well. This secondary antibody often has an
enzyme attached to it
A substrate for this enzyme is then added. Often, this substrate changes
colour upon reaction with the enzyme. The colour change shows that
secondary antibody has bound to primary antibody, which strongly implies
that the donor has had an immune reaction to the test antigen.
The higher the concentration of the primary antibody that was present in
the serum, the stronger the colour change. Often a spectrometer is used to
give quantitative values for colour strength
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Sandwich ELISA
The term sandwich comes from the fact that antigens are sandwiched between
detecting antibody both on the solid phase and acting as an enzyme labeled
conjugate. The antibody attaching to solid phase is sometimes called-Capture
antibody, the detecting antibody-Detector.
The sandwich systems vary depending on the source of the antibodies used. To
simplify terminology, the following definitions will be used.
Sandwich Direct
A. The detecting antibody is labelled (conjugate)
B. The capture and detecting antibodies can be from the same sample
C. The detecting antibody can be from different species
Sandwich Indirect
A. The detecting antibody is NOT labelled
B. The detecting antibody is not prepared in same species as capture antibody
C. The detecting antibody is detected using an anti-species conjugate
There are complications using the same specificities outlined after the overviews.
Sandwich Direct Sandwich Indirect
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This involves the passive attachment of antibodies to the solid phase. These
antibodies then bind antigen(s). The antigen(s) are diluted in a blocking buffer to
avoid non-specific attachment to the solid phase. Here the components of the
blocking buffer should not contain any antigens which might bind to the capture
antibodies.
After incubation and washing an antibody-antigen complex is attached to the solid
phase.
The captured antigen (sometimes referred to as "trapped"), is then detected by
addition and incubation of enzyme labelled specific antibodies in blocking buffer
Thus, this is a direct conjugate binding with the antigenic targets on the captured
antigen.
This second antibody can be the same as that used for capture, or be different in
terms of specific animal source or species in which it was produced.
After incubation and washing,the bound enzyme is developed by the addition of
substrate/chromogen and then stopped and read using a spectrophotometer.
• Since a single enzyme-conjugated antibody is used, the system is limited to the
specificities and properties inherent in that particular antibody 'set'. This limits
the versatility of the test, e.g., each antibody preparation used must be labelled
(for different antigens), in the same way as the Direct ELISA was limited to single
antibody preparations.
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• The system also is limited in that antigens have to have at least two antigenic
sites, since both the capture and the detecting antibodies need to bind. This can
limit the assay to relatively large antigenic complexes.
• The capture antibody (on the solid phase) and the detecting antibody, can be
against different epitopes on an antigen complex. This can be helpful in orienting
the antigenic molecules so that there is an increased chance that the detecting
antibodies will bind. It can also be an advantage when investigating small
differences between antigenic preparations by use of different detecting
antibodies and a common capture antibody.
• More versatile and hence appropriate systems are dealt with below (Sandwich
ELISA-Indirect), for this purpose. The use of exactly the same antibodies for
capture and detection (e.g., use of monoclonal antibodies) can lead to problems
whereby there is a severe limitation of available binding sites for the detector.
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This has similar steps to the Sandwich Direct for coating with capture antibody
and addition of antigens. Thus, antibodies are passively attached to the solid phase
and antigen(s) are captured.
However, the detection of the antigen involves the addition of unlabelled
antibodies.
After incubation and washing, the detecting antibodies are themselves 'detected'
by addition and incubation with an anti-species enzyme conjugate.
The bound conjugate is then processed as described in the other systems.
The advantage of this assay is that any number of samplescontaining antibodies can
be added to the captured Ag, provided that the species in which it was produced is
not the same as the capture Ab. More specifically, that the enzyme conjugated
anti-species antibody, does not react with the antibodies used to capture the
antigen.
It is possible to use the same species of antibody, if immunochemical techniques
are used to select and produce particular forms of antibodies and with attention to
the specificity of the enzyme conjugate used.
Thus, as an example, the capture antibody could be processed to a bivalent
molecule without the Fc portion (a so called FAb2 fraction). The detecting
antibodies could be untreated. The enzyme conjugate could then be an anti-species
anti-Fc portion of the immunoglobulin molecule. Thus, the conjugate would only
react with antibodies containing Fc (and not therefore the capture molecules).
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The need to devise such assays depends on the reagents available. It may be that a
monoclonal antibody is available that confers a desired specificity as compared to
polyclonal sera or that one wishes to screen a large number of monoclonal
antibodies against an antigen which must be captured (it may be at low
concentration or in a mixture of other antigens). In this case use of polyclonal sera
is unsuccessful, therefore the preparation of FAb2 fragments for the capture
antibody is worthwhile, and in facts relatively easy to use kits are available for this
purpose. The use of a commercially available anti-mouse Fc completes the
requirements.
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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).
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Immunometric Assays
Numerous schemes have been developed that use antibodies to capture and
measure analytes. The details of some can be complex, but most are designed
around two basic strategies. Perhaps the easiest to understand is the
immunometric assay (Figure 3):
• Antibodies immobilized onto a plastic surface (most often a 96-well microtiter
plate) are used to capture the target antigen present in the sample.
• A second antibody linked to an enzyme (the conjugate) is then added. It binds to
a different location on the target antigen.
• Plate wells are washed to remove unbound components.
• Substrate is added. Bound enzyme present reacts with the substrate, yielding
color.
• The enzymatic reaction is stopped in order to establish a consistent time period
for all wells. After stopping, the color is measured.
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Immunometric assays are also commonly used to measure antibodies as the analyte
(Figure 5). In this case a capture antigen is fixed to the plastic surface, and the target
antibody binds to it. An antibody-enzyme conjugate that binds to the target antibody is
then added, the plate is washed, and the conjugate reacts with the substrate to produce
the color.
Because the analyte in an immunometric assay is surrounded on two sides, the
procedure is often referred to as a sandwich assay. The acronym ELISA (Enzyme
Linked Immuno Sorbent Assay) is also often associated with sandwich assays, but
some authorities prefer to use the term in a more general sense for all sorts of
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microtiter plate immunoassays that involve enzymatic labels. The acronym IEMA is
used more formally to refer to the Immunoenzymometric assay.
Advantages of EIA
Specific and sensitive assays of wide applicability
Equipment required is relatively cheap and is widely
available
Reagents are relatively cheap and have a long shelf-life
Manipulations are simple
Assays may be very rapid
A separation step may not be required
The variety of labels available may allow multiple,
simultaneous assays to be performed
Potential for automation
No radiation hazards
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An example of an ELISA experiment
The sample is added to plate in duplicate or triplicate and then the mean
result is calculated
The quality control sample which is provided with the kit is treated as the
test samples
After reading the results the standard curve is drawn were the
concentration is blotted on the X-axis and the absorbance on the Y-axis
The standards concentrations is specified on the x-axis and the reading of
each standard is specified on the y-axis and the standard curve is drawn
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Applications for Immunoassays:
Tumor Markers, e.g. AFP, CEA, hCG, PSA …
Cardiac Markers, e.g. CK-MB, CRP, Digoxin, Myoglobin …
Cell based Assays, e.g. cell cytotoxicity …
Allergy, e.g. histamines, egg, milk, allmonds …
Growth Deficiency, e.g. hGH
Enzyme acivity
Hormone and Steroid Screening, e.g. T4, fT3, TSH …
Drug Abuse Screening, e.g. amphetamines, cocaine, LSD …
Immunological Screening
Infectious Diseases, e.g. Chlamydia, CMV, Hepatitis, Rubella …
Veterinary, e.g. bacterial infection, fertility, drugs, BSE …
Food and Beverages, e.g. pathogens, toxins…
Water Analysis, e.g. bacterial contamination, toxins, heavy metalls …
Agriculture, e.g. endotoxins, pesticides …
Environment, e.g. industrial chemicals, pesticides, surfactants …