Enzyme Immunoassays Overview

Labelling Techniques

• Labelling techniques are used in immunoassays to detect antigens or antibodies that may be small or present in low concentrations.
• These techniques use labelled reactants to determine if specific binding has occurred between the antigen and antibody.

Labelled Immunoassays

• Designed for antigens and antibodies that may be small in size or present in very low concentrations.
• Presence of antigens or antibodies is determined indirectly using a labelled reactant to detect binding.

Formats of Labelled Immunoassays: Competitive Immunoassays

• All reactants are mixed simultaneously.
• Labeled antigen competes with unlabeled patient antigen for a limited number of antibody binding sites.
• The amount of bound label is inversely proportional to the concentration of the labeled antigen. More labeled antigen detected means less patient antigen is present.

Formats of Labelled Immunoassays: Non-Competitive Immunoassays

• An antibody (capture antibody) is first passively absorbed to a solid phase (e.g., microtiter plates, nitrocellulose membranes, or plastic beads).
• Excess antibody is present to capture any patient antigen present.
• Unknown patient antigen is added and allowed to react with the solid-phase antibody.
• Unbound antigen is removed by washing, followed by adding a second antibody with a label.

Formats of Labelled Immunoassays: Non-Competitive Immunoassays - continued

• In this assay, the amount of label measured is directly proportional to the amount of patient antigen.
• This type of assay is generally more sensitive than competitive immunoassays.
• The label must not alter the molecule’s reactivity and should remain stable during the reagent's shelf life.

Heterogeneous vs Homogeneous

• Immunoassays are categorized based on whether separating bound and free reactants is necessary.
• Heterogeneous assays require a separate step to physically separate bound from free analyte.
• Homogeneous assays do not require this separation step, they are less sensitive.

Radioimmunoassay (RIA)

• First developed immunoassay. Pioneered by Yalow and Berson in the 1950s.
• Useful for hormones, vitamins, and serum proteins.
• Uses radioactive substance as a label. Radioactive elements decay spontaneously, emitting matter and energy.
• Iodine-125 (125I) is a commonly used radioactive label.

Enzyme Immunoassay (EIA)

• Enzymes are used as labels (e.g., Horseradish Peroxidase, Alkaline phosphatase, and β-D-galactosidase).
• Enzymes react with substrates to produce breakdown products.
• These products may be chromogenic (colour-producing), fluorogenic (producing fluorescence), or luminescent (emitting light).
• Commonly used in clinical lab. due to sensitivity, low cost, specificity and simplicity.

Formats of EIA - Competitive EIA

• The first enzyme immunoassays were competitive assays based on the principles of RIA.
• Enzyme-labeled antigen competes with unlabeled patient antigen for a limited number of antibody binding sites on a solid phase.
• Used generally to measuring small, relatively pure antigens like drugs or hormones.

Formats of EIA - Non-Competitive EIA

• Most noncompetitive assays are indirect (ELISAs), where the enzyme-labeled reagent doesn't participate in the initial antigen-antibody binding reaction.
• The enzyme-labeled reagent binds after the initial antigen-antibody reaction.
• Widely used in clinical labs for its sensitivity, specificity, simplicity, and low cost.
• Antigen is typically bound to a solid phase (e.g., microtiter plates, nitrocellulose membranes, or magnetic beads).
• Patient serum or a sample is added and allowed to react, and an enzyme-labeled secondary antibody is added after a wash.

Formats of EIA - Sandwich/Capture Assay

• If antibody is bound to a solid phase, these assays are called sandwich immunoassays or capture assays
• The assay must have multiple epitopes.
• Excess antibody on solid phase combines with sample to capture present antigen.
• After incubation, an enzyme-labeled antibody is added that binds to a different epitope.
• Enzymatic activity is directly proportional to the amount of antigen in the sample and can detect multiple determinants such as antibodies, cytokines, proteins, tumour markers, and microorganisms (especially viruses).

Homogenous EIA

• Homogeneous enzyme immunoassays are generally less sensitive.
• No washing or separation steps are needed.
• Rapid, simple, and easy to automate.
• Primarily used for determining low-molecular-weight analytes in serum or urine (e.g., hormones, therapeutic drugs, and drugs of abuse),

Fluorescent Immunoassay (FIA)

• Fluorescent tags or labels on antibodies are used to localize antigen in cells or tissues when bound to antigen
• Detected under UV light using a fluorescent microscope
• Direct or indirect techniques exist.

Formats of FIA - Direct Immunofluorescence

• Unlabeled fluorescent-tagged antibody is used
• Directly binds to unknown antigen fixed on a slide to create a visible fluorescent signal (green, orange-yellow) which shows up as bright objects against the dark background.

Formats of FIA - Indirect Immunofluorescence

• Patient serum is first incubated with a known antigen on a slide
• Then washed, followed by fluorescently conjugated anti-human immunoglobulin.
• This creates a fluorescence sandwich, with fluorescence localization directly proportional to antibody presence.

Fluorescence Polarization Immunoassay (FPIA)

• Based on changes in polarization of fluorescence emitted from a labelled molecule upon antibody binding.
• Labeled antigens compete with unlabeled antigen in the sample for antibody binding sites.
• More antigen present means less fluorescence polarization detected.

Chemiluminescent Immunoassays (CLIA)

• Another method to visualize antigen-antibody combination.
• Involves an oxidation reaction that produces excited molecules. Molecules then decay back to their original state, emitting light.
• Luminol, acridinium esters, ruthenium derivatives, or nitrophenyl oxalates are commonly used in these assays.

Rapid Immunoassays

• Membrane-based, easy to perform, and reproducible.
• Designed for point-of-care or home testing but can be modified for increased clinical sensitivity/semiquantification.
• Immunochromatography combines the steps into a single one-step method.