Key Notes on Labeled Immunoassays

  • Learning Outcomes:

    • Appreciate differences between competitive vs noncompetitive immunoassays.
    • Learn heterogeneous vs homogeneous immunoassays.
    • Understand labels: colorimetric, chemiluminescent, radioactive, fluorescent.
    • Comprehend competitive binding principles in immunoassays.
    • Recognize common enzymes used in enzyme immunoassays (EIAs).
    • Learn biotin-streptavidin labeling principles.
    • Outline steps for noncompetitive indirect ELISA and sandwich assays.
    • Discuss clinical applications for homogeneous EIAs and rapid immunoassays.
    • Compare analytical sensitivities of different labels.

  • Immunoassay Formats:

    • Heterogeneous Immunoassays: Requires physical separation of bound/free components (e.g., ELISA).
    • Homogeneous Immunoassays: No separation needed, quicker results (e.g., EMIT).

  • Competitive vs Noncompetitive Immunoassays:

    • Competitive: Labeled antigen competes with patient antigen for antibody binding sites. Inverse proportionality of label detected to analyte concentration.
    • Noncompetitive: Patient antigen is captured by solid-phase antibody before adding labeled antibody. Direct proportionality of label detected to analyte concentration.

  • Labels in EIAs:

    • Common Enzymes: HRP, ALP, G6PDH.
    • Sensitivity Order: Chemiluminescent > Fluorescent > Colorimetric.

  • Biotin-Avidin System: Biotin enhances sensitivity when used with streptavidin in assays.

  • Interferences in Immunoassays:

    • High-dose hook effect: Excess antigen can lead to falsely low results.
    • Antibody Interference: Autoantibodies can create false positives.
    • Biotin Interference: High biotin levels may cause erroneous results.
    • Cross-Reactivity: Leads to detection of substances other than the analyte, often resulting in false positives.

  • Key Applications:

    • Rapid Immunoassays: Used in point-of-care settings (e.g., pregnancy tests).
    • Multiplex Immunoassays: Allows simultaneous detection of multiple antibodies/antigens for high-throughput testing.

  • Summary: Understand underlying principles and classifications in immunoassays; recognize their applications and interferences.