Immunoassays: Labeled vs. Unlabeled

Unlabeled vs. Labeled Immunoassays

• Unlabeled immunoassays: simple techniques often done without sophisticated equipment.
• Labeled immunoassays: designed to improve analytical sensitivity, allowing detection of substances at lower concentrations.

Analytes in Immunoassays

• Analytes are substances measured, including antigens and antibodies found in blood, urine, and tissues.
• Biological analytes include proteins, peptides, hormones, tumor markers, immunoglobulins, and microbial antigens.
• Haptens (small-molecule antigens) like steroid hormones or drugs can be measured.
• Detecting antibody levels is useful for clinical diagnoses.

Analyte Properties

• The analyte of interest is generally a protein that can be either an antigen or antibody.
• Anti-human immunoglobulin reacts with the analyte if it's an antibody in patient serum and is derived from another species (mouse, rabbit, goat).
• One reactant (antigen or antibody) contains a label for monitoring binding.

Affinity, Avidity, and Specificity

• Affinity: Initial attraction between a single antigen-binding site on an antibody and a single epitope.
• Avidity: Overall strength of antigen-antibody binding due to the sum of binding affinities between multiple sites.
• High analytical specificity: Only the analyte of interest is detected and measured.
• Analytical sensitivity: Lowest measurable concentration of analyte, defined by the lower limit of detection (LoD) and influenced by the label and antigen-antibody affinity/avidity.

Sensitivity Optimization

• Optimal analytical sensitivity is achieved with a chemiluminescent substrate, followed by a fluorophore.
• Colorimetric products are the least sensitive.
• Radioisotopes have sensitivity close to fluorescent labels.
• Any label can be coupled to any assay design.

Immunoassay Labels

• Labeled immunoassays include a detection molecule (label).
• Current techniques use non-isotopic labels for light signal generation, such as colorimetric substrates, fluorescent compounds (fluorophores), or luminescent molecules.
• Early immunoassays used radioactive isotopes, now less common.
• Labels serve as "tracers" for detection signal tracing.
• Enzyme-mediated catalysis of a reagent substrate is a common technique to generate a light signal.

Types of Immunoassays

• Two major formats exist for labeled immunoassays: heterogeneous and homogeneous.
• Separation of bound and free tracer label is needed in all designs.
• Heterogeneous immunoassays: Require physical separation of bound and free components using methods like centrifugation, solid-phase binding, or magnetic separation; solid-phase materials include polystyrene wells, microparticle beads, latex beads, and plastic tubes.
• Homogeneous immunoassays: Do not require a physical separation step, but the detection signal and patient analyte concentration measurement methods vary.

Competitive Immunoassays

• Reagents consist of limited antibody (Ab), tracer or labeled antigen (Ag*), and reagent substrate.
• Patient antigen (Ag) concentration is the variable of interest.
• All reactants are mixed simultaneously, where labeled tracer antigen (Ag*) competes with unlabeled patient antigen (Ag) for limited antibody (Ab) binding sites.
• The amount of antibody-bound label (Ag*Ab) is measured after washing away unbound label.
• If patient antigen is present, it occupies binding sites, decreasing the amount of bound label detected.
• Bound label amount is inversely proportional to the labeled antigen concentration; more labeled antigen detected means less antigen in the patient sample.

Noncompetitive Immunoassays

• Reagent antibody (capture antibody) is passively absorbed onto a solid-phase material.
• Excess capture antibody is used to ensure patient antigen binding.
• Patient antigen reacts with the solid-phase antibody.
• A labeled antibody, directed against a different antigen epitope, is added after washing.
• Measured label amount is directly proportional to the patient antigen amount.

Radioimmunoassay (RIA)

• Pioneered by Yalow and Berson, it was the original immunoassay using a radioisotope label, measuring insulin in humans.

Enzyme Immunoassays (EIAs)

• EIAs use enzymes as labels, serving as alternatives to RIA; they catalyze biochemical reactions to produce detectable products like colored or UV light, fluorescence, and luminescence.
• Addition of an enzyme label improves analytical sensitivity.
• Enzyme properties, ease/speed of detection, and stability guide enzyme selection like horseradish peroxidase (HRP) and alkaline phosphatase (ALP).
• EIAs can be either heterogeneous or homogeneous.

Competitive Enzyme Immunoassays

• Developed by Eva Engvall and Peter Perlmann in 1971, capture antibodies are adsorbed onto plastic tubes.
• Alkaline phosphatase (enzyme) is conjugated to the antigen of interest.
• Sample antigen competes with enzyme-conjugated antigen for antibody binding sites on a solid phase.
• Enzyme activity converts the substrate into a detectable product, where product signal is inversely proportional to the test substance concentration.

Noncompetitive Enzyme Immunoassays

• ELISA/EIA methodology can be based on a noncompetitive design, used to detect antigens or antibodies.
• Indirect ELISAs detect antibodies, while "capture immunoassays" detect antigens.

Indirect ELISAs

• Used to detect a patient antibody and are termed "indirect" because the enzyme-labeled reagent is the secondary antibody and doesn't involve in the initial antigen-antibody reaction.
• Clinical applications include detecting antibody production to infectious agents difficult to isolate and detecting autoantibody production.

Capture (Sandwich) Immunoassays

• Also known as sandwich or immunometric assays
• Used to detect antigens with multiple epitopes via antibody bound to a solid-phase.
• The use of monoclonal antibodies enables a sensitive test system.

Biotin-Avidin Labeling

• Improves analytical sensitivity of labeled immunoassays by complexing biotin to the capture antibody and streptavidin to the solid-phase.
• The signal for non-isotopic labels are amplified.

Interferences in Immunoassays

• False positives or negatives results can be produced simply because of the specimen itself, that may cause quenching of light emission or exhibit background fluorescence.
• Additional test interferences may occur, especially when heterogeneous immunoassay design methods are used.

Antigen Interference

• Also known as the high-dose hook effect or postzone effect, is when excess patient antigen causes falsely decreased detection, leading to a low or normal analyte concentration.

Antibody Interference

• Whenever the antibody produced is similar to that of the test kit reagent, antibodies produced in vivo may interfere with immunoassays.

Rheumatoid Factor (RF)

• RF is an antibody directed against the Fc portion of IgG molecules.
• It can bind to IgG molecules in immunoassays, which produces false-positive results.

Heterophile Antibodies

• Heterophile antibodies react with animal proteins.
• It produce false decreases or increases by linking the capture and detection antibodies or binding to the antigen of interest.

Human Anti-Mouse Antibodies (HAMAs)

• A type of heterophile antibody, commonly causes interference in immunoassays.

Biotin Interference

• An unpredictable interference that occur when test reagent kits also use a biotin-SAv complex.
• Reported test results are found to be inconsistent with the clinical picture.

Other Technical Concerns

• Cross-reactivity is the detection of a substance other than the analyte of interest, it occurs when the substances are similar structures.
• The reference interval for an immunoassay can vary widely across manufacturers.

Homogeneous Enzyme Immunoassays

• Used for low-molecular-weight analytes (haptens), providing quicker test results with fewer analytical interferences.

Enzyme-Multiplied Immunoassay Technique (EMIT)

• The EMIT immunoassay is based on the principle of change in enzyme activity as specific antigen-antibody interaction occurs in solution.
• Free analyte competes with enzyme-labeled analyte for antibody-binding sites.

Cloned-Enzyme Donor Immunoassay (CEDIA)

• CEDIA uses genetic engineering techniques for enzymatic activity and signal that are directly proportional to patient Ag concentration.

Chemiluminescent Immunoassays

• Most immunometric assays today use chemiluminescent label detection.
• Light emission is the results from chemical reactions, where substances are oxidized.

Chemiluminescent Technology

• Detects antigens & antibodies and can be applied to heterogeneous or homogeneous immunoassay formats.

Chemiluminescent Microparticle Immunoassay (CMIA)

• Uses antibody-coated microparticles and are useful for drug measurements.

Electrochemiluminescence Immunoassay

• Uses Ruthenium and undergoes an electrochemiluminescent reaction to give of light for measurements.

Fluorescent Immunoassays

• Antibodies can be labeled so that fluorescent compounds, called fluorophores or fluorochromes, can absorb energy from an incident light source and convert that energy into light.

Fluorescent-Labeled Antibodies/Antigens

• Fluorescent-labeled antibodies or antigens have been used in a variety of immunoassays, consisting of direct and indirect immunofluorescence assays, multiplex immunoassays (MIAs), and fluorescence polarization immunoassays (FPIAs).

Direct Immunofluorescence Assays

• Used to detect antigens on tissue sections or in live cell suspensions, where fluorescent probe is detected under UV light,

Cell-Based IFA Methods

• Combines principles of hematology cell counters with fluorescent-labeled antibodies to better classify cells, such as T and B lymphocytes.

Direct vs. Indirect Immunofluorescence Assays

• Patient antigen is fixed to a microscope slide and incubated directly with a fluorescent-labeled antibody
• Well-characterized tissues or cells are fixed to slides and the anti-immunoglobulin is added. After a second wash step to remove any uncombined anti-immunoglobulin, the fluorescence of the sample is determined.

Indirect Immunofluorescence Assays

• Can be used for either antigen or antibody identification. IIF assays involve two steps.

Multiplex Immunoassay (MIA)

• A fluorescent labeling and detection platform that detects multiple antibodies simultaneously for autoimmune.

Fluorescence Polarization Immunoassays (FPIA)

• Based on a competitive design, where labeled antigen competes with patient sample antigen for a limited number of antibody-binding sites.

Rapid Immunoassays

• Rapid tests used for point-of-care testing that detect hCG and drugs in urine.