Labeled Immunoassays - Lecture Notes

Labeled Immunoassays

Chapter Overview

  • Introduction to labeled immunoassays.

  • Comparison between heterogeneous and homogeneous assays.

  • Distinction between competitive and noncompetitive immunoassays.

  • Overview of radioimmunoassay (RIA) and enzyme immunoassays (EIAs).

  • Discussion of interferences that may affect EIAs.

  • Exploration of chemiluminescent assays and fluorescent immunoassays.

  • Introduction to rapid immunoassays.

Labeled Immunoassays

  • Designed for detecting antigens and antibodies which may be:

    • Small in size.

    • Present in very low concentrations.

  • Utilizes a reactant labeled with a detection molecule to monitor the amount of specific binding that has occurred.

  • Common analytes detected include:

    • Microbial antigens.

    • Hormones.

    • Drugs.

    • Tumor markers.

    • Specific immunoglobulins.

Immunoassay Labels

  • Various types of labels that can be used in immunoassays:

    • Enzyme/colorimetric substrate: Enzymes react with substrates to produce a measurable product.

    • Chemiluminescent molecule/trigger solution: Light emitted during a chemical reaction indicates the presence of an analyte.

    • Fluorescent compound (fluorophore): Fluorochromes absorb and emit light to signal the presence of target molecules.

    • Radioactive isotope (older methods): Utilized in radioimmunoassays for detecting trace analytes.

Heterogeneous vs. Homogeneous Immunoassays

  • Heterogeneous Immunoassays:

    • Involve a physical separation of bound and free components post-reaction.

    • Commonly utilize solid-phase binding techniques:

    • Polystyrene reaction wells.

    • Microparticle beads.

    • Latex beads.

    • Plastic tubes.

    • Magnetic separation or centrifugation can facilitate this process.

  • Homogeneous Immunoassays:

    • Do not require any physical separation step after the reaction.

Competitive Immunoassays

  • All reactants mixed together at once, allowing for competition for binding sites on antibodies.

  • Mechanism:

    • Labeled and unlabeled antigen compete for a limited number of binding sites on reagent antibody.

    • Amount of bound label is inversely proportional to the concentration of the labeled antigen.

  • Characteristics:

    • Highly specific, suitable for measuring small antigens that are relatively pure (e.g., drugs and hormones).

  • High specificity allows detection of minimal antigen concentrations.

Competitive Immunoassay Principle

  1. Unknown concentration of analyte in a patient sample (represented by red dots) competes with labeled analyte (represented by yellow stars) for binding to immobilized antibody.

  2. A wash step is performed to remove unbound materials.

  3. After a substrate is added, a colored product (signal) is formed, with intensity proportional to the amount of enzyme-labeled analyte bound to the antibody.

  4. Signal strength is inversely related to analyte concentration, leading to:

    Signal representation

Noncompetitive Immunoassays

  • Known as capture, sandwich, or immunometric assays.

  • Mechanism:

    1. Patient antigen is captured by antibody bound to a solid phase.

    2. A washing step removes unbound antigen.

    3. Enzyme-labeled antibody is introduced for detection.

    4. The amount of signal generated is directly proportional to the amount of antigen present in the sample.

Noncompetitive Immunoassay Principle

  1. Reagent antigen immobilized on solid phase captures antibody in patient sample.

  2. Wash to remove unbound materials.

  3. Add enzyme-labeled detection antibody for binding.

  4. Another wash is performed to eliminate unbound antibody.

  5. Substrate is added, and the signal (e.g., color intensity) is measured:

    • Signal directly proportional to antibody concentration in the patient sample.

      Analyte representation

Radioimmunoassay (RIA)

  • The first developed immunoassay.

  • Utilizes radioactive labels such as ^{125}I, which emits gamma radiation detected by a gamma counter.

  • Characteristics:

    • Extremely sensitive and precise.

    • Suitable for measuring trace amounts of analytes like hormones, serum proteins, and drugs.

  • Competitive assay structure:

    • The amount of label in the bound phase is inversely proportional to the amount of patient antigen present.

  • Disadvantages include:

    • Handling radioactive substances (health hazard).

    • Disposal of low-level radioactive waste.

    • Short shelf life for certain reagents, limiting clinical lab testing.

Enzyme Immunoassays (EIAs)

  • Highly sensitive assays utilizing enzymes as detection labels.

  • Enzymes react with substrates to produce measurable products (chromogenic, fluorogenic, or luminescent).

  • Available formats:

    • Competitive

    • Noncompetitive

  • Common enzymes used include:

    • Alkaline phosphatase

    • Horseradish peroxidase

    • Glucose-6-phosphate dehydrogenase (G6PDH)

    • β-D-galactosidase

Indirect ELISA

  • Enzyme-linked immunosorbent assay (ELISA) used as a noncompetitive immunoassay to detect antibodies in patient samples, including:

    • Antibodies to infectious agents (e.g., hepatitis B, rubella virus).

    • Autoantibodies (e.g., antinuclear antibodies, thyroglobulin antibody).

Indirect ELISA Principle

  • Commonly automated, offering high sensitivity, specificity, ease of use, and low cost.

Capture (Sandwich) Immunoassays

  • Used to detect antigens in patient samples, particularly those with multiple determinants (e.g., cytokines, proteins, tumor markers).

  • Mechanism:

    1. Antigen in test sample binds to a solid-phase antibody.

    2. After incubation, an enzyme-labeled antibody binds to complete the sandwich formation.

    3. Addition of substrate yields a colored or chemiluminescent product detected, with enzyme activity directly proportional to the antigen amount in the sample.

Biotin-Avidin Labeling

  • Biotin: Also known as Vitamin B7 (Vitamin H).

  • Streptavidin (SAv): A bacterial protein with high affinity for biotin.

  • Biotin can be complexed to antibodies and streptavidin attached to solid-phase materials for enhanced signaling and sensitivity in ELISAs and capture immunoassays.

Interferences with Immunoassays

  • Interferences can arise from:

    • Specimen properties.

    • Antigen interference.

    • Antibody interference.

  • Note: High-dose biotin supplements can lead to false results in assays using biotin-SAv labeling.

  • Results affected can be either false-positive or false-negative.

High-Dose Hook Effect

  • Occurs when excess patient antigen leads to falsely decreased detection. Analyte concentrations may appear low or normal when they are actually high.

Antibody Interferences

  • Autoantibodies (e.g., rheumatoid factor) may cause a false positive.

  • Heterophile antibodies generally lead to false-positive results (e.g., Human and mouse antibodies - HAMA).

Homogeneous EIAs

  • Generally exhibit lower sensitivity than heterogeneous assays but are rapid and easy to perform.

  • Includes techniques like EMIT and CEDIA, often used for analyzing low-molecular-weight analytes in:

    • Serum and urine.

    • Hormones.

    • Therapeutic drugs.

    • Drugs of abuse.

  • No washing or separation step needed; enzyme activity correlates directly with patient antigen or hapten concentration:

    • When antibody binds to specific determinant sites on antigen, the active site's enzyme is blocked, leading to measurable loss of activity.

General Principle of a Homogeneous Immunoassay

  • Reagent antibodies are in solution; patient antigen and enzyme-labeled antigen are introduced:

    • They compete for limited binding sites on antibodies.

    • When patient antigen present, the enzyme label on reagent antigen isn't blocked, leading to observable color development.

  • Example conditions:

    • Sample A with low patient antigen concentration shows minimal color.

    • Sample B with higher antigen concentration displays stronger color development.

Chemiluminescent Immunoassays

  • Highly sensitive automated assays for detecting:

    • Antigens (therapeutic drugs, steroid hormones).

    • Antibodies.

  • Involves light emission from a chemical reaction, often oxidation, resulting in an excited molecule returning to a ground state.

  • Chemiluminescent molecules include:

    • Acridinium esters.

    • Ruthenium derivatives.

    • Nitrophenol oxalates.

  • Applicable to both heterogeneous and homogeneous assays:

    • Chemiluminescent microparticle immunoassay (CMIA): Heterogeneous assay where patient antigen competes with chemiluminescent antigen for antibody-coated microparticles. Physical separation done through magnetic attraction.

    • Electrochemiluminescence immunoassay (ECLIA): Utilizes ruthenium labels reacting at an electrode surface.

Fluorescent Immunoassays

  • Utilize fluorochromes as labels that absorb energy from incident light and emit light at a longer wavelength as excited electrons return to ground state.

  • Examples of fluorescent compounds include:

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

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

Direct Immunofluorescence Assays

  • Applied to identify pathogens in patient samples.

  • Method:

    • Antibody conjugated with a fluorescent tag is introduced to tissue sections or cells on a microscope slide.

    • After incubation and wash, the slide is observed using a fluorescence microscope.

Indirect Immunofluorescence Assays

  • Patient serum incubates with microscope slides containing known antigens.

  • Following a wash, an anti-human immunoglobulin labeled with a fluorescent tag is applied.

  • This forms a sandwich with the antibody, localizing fluorescence for observation.

  • Useful for identifying patient antibodies (e.g., ANAs).

Direct vs. Indirect Immunofluorescence Assays

  • Direct Immunofluorescence: Patient antigen is fixed to a slide and incubated directly with labeled antibody. Unbound antibodies are washed away. Fluorescence indicates specific antigen presence.

  • Indirect Immunofluorescence: Fixed tissues or cells are used, with patient serum-derived antibodies binding cognate antigens. After a second wash and addition of labeled anti-human immunoglobulin, fluorescence quantifies patient antibody presence.

  • Both techniques face subjectivity issues in slide reading.

Multiplex Immunoassay (MIA)

  • Fluorescent immunoassay capable of simultaneous detection of multiple antibodies or antigens.

  • Method:

    • Patient serum incubates with polystyrene beads linked to different antigens.

    • Addition of fluorescent-tagged anti-human immunoglobulin detects antibody binding.

    • Beads are identified by flow cytometry based on unique shades of red, thus indicating bound antibodies.

Rapid Immunoassays

  • Single-use, membrane-based assays utilizing immunochromatography for quick results and ideal for point-of-care testing.

  • Procedure:

    1. Patient sample added to a test membrane.

    2. Sample combines with labeled antigen or antibody (e.g., colored latex or colloidal gold).

    3. Immune complexes migrate, forming a colored reaction.

Rapid Immunoassays: Immunochromatography

  • Steps:

    • (A) Patient sample enters a cassette with antibody labeled by colloidal gold.

    • (B) Sample combines with antibody, and capillary flow moves it along.

    • (C) Monoclonal antibody captures the patient antigen attached to the gold-labeled antibody.

    • (D) Control line captures colloidal gold-labeled antibodies, providing result clarity when testing.