ELISA: Antigen-Antibody Reaction

• ELISA is an antigen-antibody reaction.
• It is a common laboratory technique to measure the concentration of antibodies or antigens in the blood.
• ELISA is used for detecting and quantifying substances: peptides, proteins, antibodies and hormones.

ELISA Principle

• Enzyme immunoassays (EIAs) use the catalytic properties of enzymes to detect and quantify reactions.
• ELISA is a heterogeneous EIA technique used in clinical analyses.
• One of the reaction components is nonspecifically adsorbed or covalently bound to the surface of a solid phase.
• Attachment facilitates the separation of bound and free-labeled reactants.
• Specific antibodies in a sample can be quantified using an ELISA procedure.
• Antigens, instead of antibodies, bind to a solid phase.
• The second reagent is an enzyme-labeled antibody specific to the analyte antibody.
• ELISA assays detect antibodies to viruses and autoantigens in serum or whole blood.
• Enzyme conjugates coupled with substrates produce visible products.
• ELISA-type assays are used with results that can be interpreted visually.
• Assays are utilized in screening, point-of-care, and home testing applications.

Types of ELISA

• Four major types of ELISA:
  • Direct ELISA (antigen-coated plate; screening antibody)
  • Indirect ELISA (antigen-coated plate; screening antigen/antibody)
  • Sandwich ELISA (antibody-coated plate; screening antigen)
  • Competitive ELISA (screening antibody)

Direct ELISA

• In the process the primary detection antibody binds directly to the protein of interest.
• The plate is rewashed to remove any unbound antibodies.
• An enzyme, alkaline phosphatase (AP) or horseradish peroxidase (HRP), is added to the plate.
• The enzyme results in a color change.
• The color change of the sample is caused by the hydrolysis of phosphate groups from the substrate by AP or by the oxidation of substrates by HRP.
• Advantages: removing secondary antibody cross-reactivity.
• Direct ELISA is rapid compared to indirect ELISA, for fewer steps in process.
• Disadvantages: low sensitivity, due to only enzyme-linked substance to bind to antigen, high cost of reaction.

Indirect ELISA

• The process requires two antibodies: a primary detection antibody that sticks to the protein of interest, and a secondary enzyme-linked antibody complementary to the primary antibody.
• The primary antibody is added first, and followed by a wash step.
• After the wash, the enzyme-conjugated secondary antibody is added and incubated.
• Then, the ELISA steps are the same as the direct ELISA with a wash step, the addition of substrate, and the detection of a color change.
• Compared to direct ELISA: indirect ELISA has higher sensitivity and is less expensive and more flexible.
• The major disadvantage is the risk of cross-reactivity between the secondary detection antibodies.

Sandwich ELISA

• Antigen can be detected by sandwich ELISA, in the procedure antibody is coated on the microtiter.
• A sample containing antigen is added to the well and reacts with the antibody.
• An antigen-antibody complex is formed.
• Once the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added.
• The second antibody reacts with the bound antigen.
• Disadvantages are the time and expense, and the necessary use of "matched pair" (divalent/multivalent antigen) and secondary antibodies.

Competitive ELISA

• Competitive ELISA tests for the presence of an antibody specific for antigens in the test serum.
• This type of ELISA uses two specific antibodies: an enzyme-conjugated antibody and another antibody present in the serum.
• Combining both antibodies into the wells help for competition for binding to antigens.
• The presence of a color change, meaning the enzyme-conjugated antibody bound the antigens, indicating the test is negative.
• The absence of color indicates a positive test and the presence of antibodies in the test serum.
• The benefits: less sample purification, it can measure a large range of antigens in a given sample, it can be used for small antigens, and it is has low variability.
• Low specifity and cannot be used in dilute samples.

Diagnostic Tests

• Detecting the Presence of Antibodies in the Blood identifies Hepatitis A, B, C, and HIV.
• Detecting and Estimating the Levels of Tumor Markers detects Prostate-specific antigen (PSA).
• Hormone Levels may be detected and estimated like Luteinizing hormone or Follicular stimulating hormone.
• Tracking Disease Outbreaks for Cholera and HIV.
• Detecting Past Exposures can identify HIV, Lyme disease, and Hepatitis.
• Screening Donated Blood for Possible Viral Contaminants detects Anti-HIV-1/2, Anti-HCV, and HBsAg.
• Detecting Drug Abuse identifies Amphetamine and Cocaine.

Interfering Factors

• Plate Assay: the shape and quality of the wells, the material of the plate, potential pre-activation, even or uneven coating.
• Buffer: pH, contamination.
• Capture and detection antibody: incubation time, temperature, specificity, titer, affinity.
• Blocking buffer: cross-reactivity, concentration, contamination.
• Target antigen: conformation, stability, epitopes.
• Enzyme conjugate: type, concentration, function, cross-reactivity.
• Washes: contamination, frequency, volume, duration, composition.
• Substrate: quality/manufacturer.
• Detection: instrument-dependent factors.
• Reader/human error.