Serology
Friday, 11 October 2024 16:35
Enzyme-linked immunosorbent assay (ELISA)
• ELISA is an enzyme immunoassay that employs enzyme-labeled immunoreactants and an
immunoadsorbent to determine the presence and concentration of certain proteins (e.g., tumor
markers, viral proteins, drugs, antibodies) in serum
• The detection method is based on the highly specific immunologic interaction between
an antibody and its antigen (i.e., the protein of interest).
1. The antigen is fixed on a microtiter plate and bound by an enzyme-coupled antibody.
2. The enzyme catalyzes a reaction when incubated with its substrate which is chromogenic,
chemifluorescent, or chemiluminescent.
3. The intensity of the signal is directly proportional to the amount of captured antigen.
• While being highly specific and sensitive, the specificity of ELISA is lower compared to western
blot.
• There are two main types of ELISA tests:
• Direct ELISA: tests for the antigen directly
• Indirect ELISA: tests for the antibody, which indicates the presence of an antigen (indirectly)
• A sandwich ELISA is an indirect ELISA that employs two antibodies that bind to
different epitopes on the antigen of interest.
Direct ELISA
1. The patient's sample supposedly containing the protein of interest (i.e., the antigen) is added to a
well of microtiter plates with a buffered solution.
2. The specific antibody-enzyme conjugate is added to the solution.
3. A substrate for the enzyme is added
4. Spectrometry is used to detect the generated chromophore
• Higher concentration of antibodies binding to the antigen → stronger signal
• Lower concentration of antibodies binding to the antigen → weaker signal
Indirect ELISA
• Same procedure as direct ELISA, with the following exceptions:
• The antibody specific for the antigen of interest is not labeled itself and is called
primary antibody.
• The primary antibody is detected by a secondary, labeled antibody.
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Serology
Friday, 11 October 2024 16:38
Sandwich ELISA
1. A surface plate is coated with capture antibodies (not the patient's antibodies).
2. The sample is added to the coated plate where the captured antibodies bind the antigen of interest.
3. Specific (labeled) antibodies for the antigen are added. ; if the antigen is present, the antibody binds
to the antigen.
4. A substrate for the enzyme is added (color, fluorescent, or electrochemical changes are due to the
reaction between substrates and enzymes).
5. Spectrometry, fluorescence, or electrochemical studies are performed to assess for the amount
of antigens present.
Uses
• Screening for HIV antibodies (high sensitivity, low specificity)
• Testing for West Nile virus antibodies
• Detection of the following organisms:
• Mycobacterium tuberculosis
• Rotavirus (in feces)
• Hepatitis B virus
• E. coli enterotoxin (in feces)
• Detection of PSA
ELISA principle
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