Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme-Linked Immunosorbent Assay (ELISA)

• ELISA is a biochemical technique used in immunology to detect the presence of an antibody or antigen in a sample.
• The technique has three formats: competitive, sandwich (direct), and indirect.

Definitions

• Antibodies (immunoglobulins, Ig): Globulin proteins in blood, used by the immune system to identify and neutralize foreign substances (bacteria, viruses).
• Antigens: Substances that stimulate the production of antibodies when introduced into the body.
• Immunoassay: A technique using antigen-antibody binding to identify and quantify specific antigens or antibodies in a sample.
• Analyte: The substance being analyzed in immunoassays; either antibody or antigen.

Antibody Production (Polyclonal Antibodies)

• Polyclonal antibodies are produced by injecting an antigen into a mammal (e.g., mouse, rabbit, goat, sheep, horse) to elicit an immune response.
• The resulting blood contains multiple antibodies that bind to the same antigen.
• Animal selection depends on the desired antibody quantity and size.

Antibody Production (Monoclonal Antibodies)

• Monoclonal antibodies are specific for a single antigen.
• Antibody-secreting lymphocytes are isolated from the animal and fused with cancer cells (hybridomas).
• Hybridomas continuously grow and secrete the same antibody.
• These antibodies are called monoclonal antibodies.

ELISA Techniques

Competitive ELISA

• Labeled antigen competes with sample antigen for antibody binding sites.
• More sample antigen leads to a weaker signal.
• Primarily used to detect small molecules.
• Signal intensity correlates with the concentration of the analyte.

Sandwich ELISA (Direct ELISA)

• ELISA plate is coated with antibody specific to the analyte (target protein).
• Antigen in the sample is captured and binds to the antibody.
• Enzyme-linked secondary antibody is added, also specifically binding to the analyte.
• Substrate is then used, causing a colored change.
• Intensity of color change reflects analyte concentration, measuring the amount of a particular target protein or other antigen captured by the plate.
• Primarily used for the detection of larger molecules.

Indirect ELISA

• Antigen is added to each well, adhering due to charge interactions.
• A non-reacting protein is used to block any remaining uncoated surfaces.
• Serum containing antibodies is added, some of which may bind specifically to the coated antigen in the well.
• A labeled secondary antibody is added, binding to the previously bounded antibodies.
• A substrate then generates a colored product whose intensity is proportional to the concentration of the primary antibody.
• This indirectly measures the analyte (the substance of interest in the serum.)

Example: An ELISA Experiment

• The 96-well plate is appropriately labeled, and the initial wells are used to create a standard curve.
• Sample is added in duplicates or triplicates for calculations of mean results.
• Quality control samples, provided with the kit, are treated like the test samples.
• Standard concentrations are marked on the x-axis (horizontal axis); the readings for each standard are placed on the y-axis (vertical axis) of a graph to create a standard curve.
• The standard curve is used to find the unknown concentrations of the samples by finding the concentration on the x-axis that corresponds to the absorbance reading (from the experiment) on the y-axis.

Colloidal Gold-based Immunoassay Test Strip

• This technique demonstrates a lateral flow immunoassay.
• It involves sample application on a test strip.
• Results are observed based on colored reactions.

This quiz explores the Enzyme-Linked Immunosorbent Assay (ELISA), a critical technique used in immunology for antibody and antigen detection. It covers various formats of ELISA, essential definitions, and the production of polyclonal antibodies.