Transcript Enzyme Linked Immunosorbent Assay (ELISA)

Enzyme Linked Immunosorbent
Assay (ELISA)
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Enzyme Linked Immunosorbent Assay (ELISA)
Term Was Coined By Engvall and Pearlmann in 1971
Similar To RIA, Except No Radiolabel
Can Be Used To Detect Both Antibody and Antigen
Very Sensitive, pg/mL
Relies on Monoclonal Abs
• ELISA may be run in a qualitative or
quantitative format.
• ELISA results are reported as a
number using a
spectrophotometer,
spectrofluorometer, or other
optical device. This test is
determining the "cut-off" point
between a positive and negative
result.
• Unknowns that generate a signal
that is stronger than the known
sample are called "positive";
those that generate weaker signal
are called "negative.
• Most commonly, ELISAs are performed in 96well (or 384-well) usually polystyrene microtiter
plates, which will passively bind antibodies and
proteins
Advantages of ELISA
 Less costly and safest.
 Easy visualization of results with high level of accuracy.
 Specific and highly sensitive assay that can detect protein at the
picomolar to nanomolar range.
 Easily automated for performance of large numbers of tests.
 Require minimal reagents.
 Qualitative detection or Quantitative measurement of either antigen or
antibody.
 Wells can be coated with antigens or antibodies.
 Can be done by personnel with only minimal training.
Applications of ELISA
 Analysis of hormones, vitamins, metabolites,
and diagnostic markers.
 Therapeutic drug monitoring.
 Diagnostic procedures for detecting infection.
Requirements for ELISA test
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Purified antigen (if you want to detect or quantify antibody).
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Purified antibody (if you want to detect or quantify antigen).
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Standard solutions (positive and negative controls).
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Sample to be tested.
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Micro-titer plates: plastic trays with small wells in which the assay is
done.
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Wash fluid (buffer).
Enzyme-labeled antibody and enzyme substrate.
• ELISA reader (spectrophotometer) for quantitative measurements.
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Enzyme labels
• Enzyme labels are used to detect the binding of antigenantibody complex.
• It should have high specific reactivity.
• It should be easily coupled to antigen-antibody complex and
must stable.
• Enzymes used in labelling should not be normally present in
the patient samples.
• Examples of enzyme labels are Horse radish peroxidase,
Alkaline phosphatase, and Glucose oxidase.
Stages in ELISA
1. The adsorption of either antigen or antibody to the 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 binding of enzyme to the target antigen or antibody.
6. The addition of substrate (production of a visible signal).
7. The visual or spectrophotometric reading of the assay.
Types of ELISA assay
– Direct ELISA
– Indirect
– Sandwich
– Competitive
Substrate
E
Secondary antibody
labeled
Primary
antibody
Target
antigen
Ag
Capture
antibody
Sandwich ELISA
Antigen
conjugate
Target
antigen
E
Ag
Ag
Ag
Ag
Capture
antibody
Competitive ELISA