Immunoassays Lecture part 2

Overview of Immunoassays

  • Immunoassays are analytical methods based on the reactions between antigens and antibodies.

  • They are essential tools in clinical chemistry.

Types of Immunoassays

  1. Western Blotting

    • Used for analyzing proteins through gel separation followed by antibody binding.

    • Applications include infectious disease confirmation and analysis.

  2. Competitive vs Noncompetitive Immunoassays

    • Competitive Immunoassays:

      • Characterized by competition between a known labeled analyte and an unknown analyte from a sample.

      • Example: Detection of drugs of abuse (e.g., THC) in body fluids.

      • Measurement is based on the competition for binding to a specific antibody.

    • Noncompetitive Immunoassays:

      • Do not involve competition. Instead, an antibody binds directly to an unknown analyte without the presence of a labeled analogous analyte.

      • Measurement is straightforward because only the unknown analyte is present.

  3. Homogeneous vs Heterogeneous Immunoassays

    • Homogeneous Immunoassays:

      • No washing step; all components are mixed together.

      • The amount of unbound labeled analyte is measured directly, which is proportional to the concentration of the unknown analyte in the sample.

      • Example: Increased concentration of the analyte results in an increased signal (label reaction).

    • Heterogeneous Immunoassays:

      • Involves a washing step to remove unbound analytes, leaving bound analytes for measurement.

      • Generally, the measurement is inversely proportional to the sample analyte concentration (i.e., increased analyte concentration results in a decreased signal).

Key Information on Competitive Immunoassays

  • Types of Competitive Immunoassays:

    • Homogeneous Competitive Immunoassays:

      • Direct measurement of labeled unbound analyte, indicating that it is directly proportional to the sample analyte concentration.

    • Heterogeneous Competitive Immunoassays:

      • After measuring, unbound labeled analytes are washed away, and the remaining measurements indicate an inverse relationship to the analyte's concentration.

  1. Noncompetitive Immunoassays

    • Signal strength is directly proportional to the analyte concentration.

    • Commonly involves a two-site noncompetitive immunoassay, also referred to as a sandwich assay.

    • Involves a capture antibody on a solid phase and a detection antibody that binds to a second epitope of the same analyte.

Specific Types of Immunoassays

  • Enzyme-Linked Immunosorbent Assay (ELISA):

    • Acronym stands for Enzyme-Linked Immunosorbent Assay.

    • Always a noncompetitive immunoassay.

    • Used to analyze for unknown antigens/antibodies in patient samples without competition from other labeled analytes.

    • Exhibits a directly proportional relationship between signal strength and concentration of the unknown analyte.

    • Applications include:

      • Infectious disease diagnostics (HIV, hepatitis, SARS-CoV-2)

      • Measurement of tumor markers (e.g., prostate-specific antigen)

      • Hormones and cytokines

      • Therapeutic drug monitoring

General Steps Involved with ELISA

  1. Coating:

    • Antigens are immobilized on a surface (e.g., polystyrene microplate).

  2. Blocking:

    • Non-specific binding sites are blocked.

  3. Probing:

    • The sample containing unknown analytes is added.

  4. Measurement:

    • The reaction signal is based on the enzyme label and substrate, generating a measurable reaction.

Types of ELISA

  1. Direct ELISA:

    • Involves fixing the antigen to a plate and detecting it with a single labeled primary antibody.

    • Measurement occurs after adding the substrate, leading to color development if the antigen is present.

  2. Indirect ELISA:

    • Similar setup to the direct ELISA, but it includes a secondary labeled antibody that binds to the primary antibody, increasing sensitivity.

  3. Sandwich ELISA:

    • The analyte is sandwiched between two antibodies: one fixed to the plate (capture antibody) and a second (detection antibody) that is labeled.

    • This format is highly sensitive and specific, making it widely used in clinical laboratories.

  • Include references to visual aids or videos to reinforce understanding of concepts.