ELISA
Introduction
Enzyme-Linked ImmunoSorbent Assay, also called ELISA, ,is probably the most
commonly used immunological assay to detect the presence of an antibody or
an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and
plant pathology, as well as a quality control check in various industries.
Most commonly, ELISAs are performed in 96-well
(or 384-well) usually polystyrene microtiter plates,
which will passively bind antibodies and proteins.
ELISA results are reported as a number using a
spectrophotometer, spectrofluorometer, or other
optical device. The most controversial aspect of this
test is determining the "cut-off" point between a
positive and negative result.
A 96 – well microtiter plate
Unknowns that generate a signal that is stronger
than the known sample are called "positive"; those that generate weaker signal are called
"negative."
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a
simple positive or negative result for a sample. The cut-off between positive and negative
is determined by the analyst and may be statistical. In quantitative ELISA, The amount of
colored product is proportional to the amount of enzyme-linked antibody that binds,
which is directly related to the amount of antibody that was present to bind antigen or
antigen that was present to bind antibody. If known amounts of antigen or antibody are
added, a standard curve , which is typically a serial dilution of the target, can be
constructed by plotting each standard optical density (ordinate) vs. the Standard
concentrations (abscissa) on graph paper. The concentration of each unknown antigen or
antibody then determined from the standard curve.
Changes in color or fluorescence can be used as a signal which indicates the quantity of
antigen in the sample. Older ELISAs utilize chromogenic substrates,though newer assays
employ fluorogenic substrates enabling much higher sensitivity.
Advantages of ELISA
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ELISA is safest as there is no radioactive material involved as in case of RIA
which were used prior to ELISA at larger scale.
ELISA allows for easy visualization of results with high level of accuracy
Sensitive (ability to detect small amounts of antigen or antibody), nanogram
levels or lower
Specific (ability to discriminate between closely related but antigenically different
molecules)
Easily automated for performance of large numbers of tests.
Minimal reagents
Qualitative & Quantitative assays
– Qualitative  Eg. HIV testing
– Quantitative assays  Eg. Therapeutic Drug Monitoring
Greater scope : Wells can be coated with Antigens OR Antibodies
Suitable for automation high speed
Can be done by personnel with only minimal training
Applications of ELISA
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Analysis of hormones, vitamins, metabolites, diagnostic markers, i.e. ACTH,
FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins,
glucocorticoids.
Therapeutic drug monitoring: Barbiturates, morphine, dioxin,
Diagnostic procedures for detecting infection: HIV, Hepatitis A, B etc.
Requirements to do the assay
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Purified antigen (if you want to detect or quantify
antibody).
Purified antibody (if you want to detect or quantify
antigen).
Standard solutions (positive and negative controls).
Sample to be tested.
Microtiter plates: plastic trays with small wells in
which the assay is done.
Wash fluid (buffer).
Enzyme-labeled antibody and enzyme substrate.
ELISA reader (spectrophotometer) for quantitative measurements.
Enzyme labels
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Enzyme is used to detect the binding of antigen-antibody complex
Enzyme labels should have high specific reactivity
Should be easily coupled to ligands & the labelled complex must be stable
The reactivity should be retained after linking of the enzyme to the
antigen/antibody
The chosen enzymes should not be normally present in the patient samples
Examples of enzyme labels: Horse radish peroxidase, Alkaline phosphatase,
Glucose oxidase
Stages in ELISA
Common practical features of the ELISA:
1. The adsorption of antigen or antibody to the plastic solid-phase (micro-titer
plate).
2. The addition of the test sample and subsequent reagents.
3. The incubation of reactants (formation of antigen-antibody complex) .
4. The separation of bound and free reactants by washing.
5. The addition of enzyme-labeled reagent.
6. The addition of enzyme detection system (production of a visible signal).
7. The visual or spectrophotometric reading of the assay.
Types of ELISA assay
Variation of different types of ELISA assay are depend upon the labeling and signal
detection methodology. The basic approaches stay the same: fixing either antigen or
antibody and detecting antibody-antigen complex.
ELISAs for Antigen Detection
Micro-titer plate coated with antibodies; Enzyme labelled antibodies.
Sandwich ELISA
The sandwich ELISA, a less common variant of this technique, measures the amount of
antigen between two layers of antibodies - the capture antibody and the detection
antibody. The antigens to be measured must contain at least two antigenic sites, capable
of binding to the antibody, since at least two antibodies act in the sandwich. In sandwich
ELISA, the production of color indicates the quantity of antigen.
PROTOCOL
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Coat the micro- titer plate with purified antibody to the antigen.
Wash away unbound antibody and cover any sites that might nonspecifically bind
with unrelated protein.
Add sample to be tested for antigen to plate.
Incubate till antigen antibody reaction is complete.
Wash off unbound antigen.
Add enzyme-labeled specific antibody to a different epitope of the antigen to
make a "sandwich".
Incubate till antigen binds labeled antibody
Wash away unbound antibody.
Add substrate for enzyme that will be converted to a colored product; measure
color.
Color proportional to antigen in test sample.
FIGURE
Sandwich ELISA
Competitive ELISAs
A competitive ELISA provides an alternative to the direct ELISA and can measure the
amount of antigen in a sample, however it is much less sensitive. In this type of ELISA,
the antigen is labeled instead of the antibody. Unlabeled antigen and the labeled antigen
compete for binding to the capture antibody and there is an inverse relationship between
the signal obtained and the concentration of the antigen in the sample, i.e. the more
antigen the lower the signal. For example, the absence of the antigen in the sample will
result in a dark color, whereas the presence of the antigen will result in a light color or no
color as the concentration of the antigen increases.
PROTOCOL
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With purified antibody to the antigen.
Add known quantities of sample to be tested,unlabeled antigen, and antigen
labelled with enzyme to plate.
Incubate: till antigen antibody reaction is complete
Wash to remove unbound antigens
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Add substrate; incubate with enzyme until color develops; measure color in a
spectrophotometer using appropriate filter.
Colour inversely related to antigen in patient sample
ELISAs for Antibody Detection:
Microtiter plate coated with antigens; Enzyme labelled secondary-antibodies.
Direct ELISA
The direct ELISA uses the method of directly labelling antibody itself. Microwell plates
are coated with a sample containing the target antigen, and the binding of labelled
antibody is quantitated by a colorimetric, or fluorescent end-point. The direct ELISA is
relatively quick.
ROTOCOL
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Coat the microtiter plate with the target antigen
Add serum sample to be tested for antibody labelled with enzyme to plate.
Incubate till antigen antibody reaction is complete
Wash to remove unbound antibody.
Add substrate ; incubate with enzyme until color develops; measure color.
Colour proportional to antibody in test sample
Indirect ELISA
The indirect, two-step method uses a labeled secondary antibody for detection. First, a
primary antibody is incubated with the antigen. This is followed by incubation with a
labeled secondary antibody that recognizes the primary antibody. The production of color
indicates the amount of an antibody to a specific antigen.
PROTOCOL
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Coat the microtiter plate with purified antigen by letting an antigen solution sit in
the wells for 30-60 minutes. Wash away unbound antigen with buffer and cover
any sites that might nonspecifically bind antibody with unrelated protein.
Washing away unbound protein.
Add serum sample to be tested for specific antibody to plate.
Incubate to allow specific antibody to bind to the antigen.
Wash off unbound antibody.
Add anti- antibody that covalently linked with enzyme.
Incubate till anti- antibody binds to specific antibody.
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Wash off unbound antibody-enzyme complex.
Add substrate: colorless substrate that the enzyme will convert to a colored
product.
Incubate until color develops; measure color in a spectrophotometer.
The Color proportional to antibody in test sample.
FIGURE
Indirect ELISA
Capture ELISA
A capture ELISA is designed to detect a specific type of antibody, such as IgG or IgM.
ROTOCOL
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Coat microtiter plate with antibody specific for IgG or IgM.
Add test sample containing IgG or IgM to the plate.
Add Specific antigen to the plate.
Incubate: till antigen antibody reaction is complete.
Wash to remove unbound antigen.
Add secondary antibody labelled with Enzyme specific to the test antigen.
Incubate till labelled secondary antibody binds antigen-antibody complex.
Wash to remove unbound labelled secondary antibody.
Add substrate ; incubate with enzyme until color develops; measure color.
Colour proportional to IgG or IgM antibody in test sample.