Labeled Immunoassays

Labeled Immunoassays Overview

  • Labeled Immunoassays: Utilize detection molecules to monitor antigen-antibody interactions.

    • Designed for small antigens or those in low concentrations.

    • Detect various analytes: microbial antigens, hormones, drugs, tumor markers, specific immunoglobulins.

Immunoassay Labels

  • Types of Labels Used:

    • Enzyme/colorimetric substrate: Enables color changes in reactions.

    • Chemiluminescent molecule/trigger solution: Light emission measurable in immunoassays.

    • Fluorescent compounds (fluorophores): Emit light upon excitation.

    • Radioactive isotopes: Older methods using radioactivity for detection.

Types of Immunoassays

Heterogeneous Immunoassays

  • Involves physical separation of components.

    • Usually binds antigens to solid phases: polystyrene wells, microparticle or latex beads, plastic tubes.

    • Techniques like magnetic separation or centrifugation may be employed.

Homogenous Immunoassays

  • No physical separation is required.

Competitive Immunoassays

  • Process Overview:

    • All reactants mixed simultaneously.

    • Labeled vs. unlabeled antigens compete for binding sites on antibodies.

    • Bound label amount inversely proportional to labeled antigen concentration.

  • Specificity:

    • Highly specific for small and pure antigens, such as drugs and hormones.

Principle of Competitive Immunoassays

  • Steps:

    1. Analyte in the patient sample (unknown concentration) competes with labeled analyte.

    2. Wash to remove unbound materials.

    3. Addition of substrate generates a colored product proportional to enzyme-labeled analyte bound to the antibody.

    • Signal strength usually inversely related to patient analyte concentration.

Noncompetitive Immunoassays

  • Also referred to as sandwich or immunometric assays.

    • Patient antigens captured by antibodies on a solid phase.

    • After washing, a second labeled antibody is added.

    • Amount of label is directly proportional to patient antigen concentration.

Principle of Noncompetitive Immunoassays

  • Steps:

    1. Reagent antigen immobilized on a solid phase, binds patient sample antibody.

    2. Wash to remove unbound materials (this may be bypassed in one-step assays).

    3. Add enzyme-labeled detection antibody bound to human immunoglobulin.

    4. Wash again to remove unbound materials.

    5. Add substrate and measure signal; directly proportional to antibody concentration.

Radioimmunoassay (RIA)

  • First immunoassay developed, using radioactive labels (e.g., ^{125}I most common).

  • Characteristics:

    • Emits gamma radiation, detected by gamma counters.

    • Extremely sensitive and precise, suitable for trace analysis of hormones, serum proteins, drugs.

  • Disadvantages:

    • Health hazards due to radioactive substances.

    • Disposal and regulatory challenges associated with low-level radioactive waste.

    • Some reagents have a short shelf life, limiting clinical lab use.

Enzyme Immunoassays (EIAs)

  • Highly sensitive assays using enzymes as labels.

    • Produce chromogenic, fluorogenic, or luminescent products.

  • Common Enzymes:

    • Alkaline phosphatase, horseradish peroxidase, glucose-6-phosphate dehydrogenase (G6PDH), β-D-galactosidase.

Indirect ELISA

  • Noncompetitive immunoassay for antibody detection against infectious agents (e.g., hepatitis B, rubella) and autoantibodies (e.g., antinuclear antibodies).

Capture (Sandwich) Immunoassays

  • Detect specific antigens in patient samples.

    • Best suited for antigens with multiple determinants (cytokines, proteins).

  • Procedure:

    • Test antigen binds to solid-phase antibody.

    • After reaction and incubation, enzyme-labeled antibody is added to form a sandwich.

    • Subsequent addition of substrate detects a colored or chemiluminescent product.

    • Enzyme activity correlates directly with antigen amount.

Biotin-Avidin Labeling

  • Biotin (Vitamin B7) can be complexed with streptavidin (SAv), a strong biotin binding protein, enhancing detection in ELISAs and capture assays.

Interferences with Immunoassays

  • Caused by specimen properties, antigen, or antibody interference.

    • High-dose biotin supplements can lead to false results in biotin-SAv assays.

High-Dose Hook Effect

  • Excess patient antigen leads to falsely decreased assay results, appearing as low analyte concentration.

Antibody Interferences

  • Autoantibodies: E.g., rheumatoid factor leading to false positives.

  • Heterophile antibodies: Commonly result in false positives (e.g., human-mouse antibody interactions)

Homogenous EIAs

  • Generally less sensitive than heterogeneous assays.

  • Rapid and straightforward to perform.

  • Formats include EMIT and CEDIA, used for low-molecular-weight analytes—hormones, therapeutic drugs, and drugs of abuse.

    • No washing or separation step required.

General Principle of Homogenous Immunoassays

  • Reagent antibody is in solution; competition for binding sites occurs between patient antigen and enzyme-labeled antigen.

    • Observed reaction color intensity correlates with patient antigen levels.

Chemiluminescent Immunoassays

  • Highly sensitive method detecting antigens or antibodies through light emission via chemical reactions.

  • Chemiluminescent molecules include acridinium esters, ruthenium derivatives, and nitrophenol oxalates.

    • Applicable to both heterogeneous and homogenous assays.

Types of Chemiluminescent Assays

  • Chemiluminescent Microparticle Immunoassay (CMIA): Heterogeneous assay where patient antigens compete with chemiluminescent antigens for antibody-coated microparticles.

  • Electrochemiluminescence Immunoassay (ECLIA): Utilizes a ruthenium label that reacts at electrode surfaces.

Fluorescent Immunoassays

  • Use fluorochromes that absorb energy from light and emit it at a different wavelength.

  • Examples:

    • Fluorescein: Absorbs light at 490-495 nm and emits green light at 520 nm.

    • Rhodamine: Absorbs light at 550 nm and emits red light at 585 nm.

Direct Immunofluorescence Assays

  • Identify pathogens in patient samples using fluorescent-tagged antibodies.

  • Incubate with fixed tissues on slides, visualize using fluorescence microscopy.

Indirect Immunofluorescence Assays

  • Patient serum binds to known antigens on slides, followed by fluorescent-tagged anti-human immunoglobulin.

    • Forms a sandwich structure for localized fluorescence detection.

    • Can identify antibodies (e.g., antinuclear antibodies).

Direct vs. Indirect Immunofluorescence Assays

  • Direct Assay: Patient antigen fixed on slides, incubated with fluorescent antibodies.

    • Observable fluorescence indicates specific antigens.

  • Indirect Assay: Known tissues fixed on slides; patient antibodies attach, followed by labeled anti-human antibodies.

    • Fluorescent intensity indicates patient antibody concentration.

Multiplex Immunoassay (MIA)

  • Allows simultaneous detection of multiple antibodies or antigens.

  • Uses polystyrene beads conjugated to different antigens; flow cytometry identifies beads with unique fluorescence.

Rapid Immunoassays

  • Single-use, membrane-based assays employing immunochromatography.

    • Fast and user-friendly, suited for point-of-care testing.

  • Patient samples combine with labeled antigens or antibodies; immune complexes migrate to form visible reactions on membranes.

Rapid Immunoassay via Immunochromatography

  • Process Overview:

    1. Patient sample added to a cassette with colloidal gold-labeled antibodies.

    2. Sample interacts with antibody and is propelled along due to capillary flow.

    3. Monoclonal antibodies capture patient antigens, forming visible lines on the test strip.