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Introduction to Immunoassays

• Immunoassays use immunochemical methods to exploit the properties of antibody-antigen (Ab-Ag) complexes
• Antibodies (Abs) are used as reagents to detect and quantify antigens (Ags)
• A wide range of compounds, from small molecules to large proteins, can be measured using immunoassays

Learning Objectives

• Describe the characteristics of homogenous and heterogeneous assays
• Outline the principles of non-competitive and competitive immunoassays
• Understand the meaning of conjugation
• List components of immunoassays that can be conjugated to antibodies and antigens

Immunoassay Design

• Careful selection of antibodies (Abs) with appropriate binding strength and specificity is crucial
• Abs should exhibit high affinity and high specificity for the target antigen
• Choosing the correct label and detection method is essential for measuring Ab:Ag binding
• Labelled Abs and Ag complexes are combined through covalent attachments (conjugation)
• Enzyme-substrate interactions generate signals that are detected
• Signal detection is a critical step in immunoassay design

Homogenous vs. Heterogenous Assays

• Homogenous immunoassays: Do not require separation steps before analysis; reagents are added simultaneously; can be competitive or non-competitive
• Heterogenous immunoassays: Require separation steps (e.g., filtration, washing, blocking) before analysis; typically multiple steps; can be competitive or non-competitive; are the most common type of immunoassay

Heterogenous Assay Characteristics

• Can be either competitive or non-competitive
• Immuno-reagents are attached to a solid phase (e.g., agarose, polystyrene beads, microtiter plates) via covalent or non-covalent (adsorption) methods
• Factors to consider during heterogeneous assay design include:
  • Immunoreaction or Ab-Ag binding should not affect signal generation
  • The enzyme label should retain its activity after the immunoreaction
  • Non-specific adsorption of components can affect the results
  • Solid phase material and edge effects can impact the results

Non-competitive Immunoassays

• An excess of unlabelled antibody captures the analyte from the sample
• A labelled secondary antibody binds to the captured analyte forming a 'sandwich' complex
• The signal generated, measured spectrophotometrically, is directly proportional to the analyte concentration
• Non-competitive techniques are generally more sensitive and reproducible than competitive assays

Non-competitive Immunoassays (Sandwich ELISA)

• Unlabeled capture antibody is immobilzed on a solid surface
• Sample (analyte) is added
• Capture antibody binds to its target antigen within the sample
• Detection antibody (linked to an enzyme) is added, binding to the second region of the antigen to form a 'sandwich'
• An enzyme substrate is added to generate a signal (colorimetric changes are commonly measured spectrophotometrically)
• Wash steps are done to remove unbound components

Non-competitive Immunoassays: Advantages and Limitations

• Advantages: Excellent detection in complex samples such as plasma and serum; high specificity and sensitivity
• Limitations: Requires carefully matched antibody pairs; difficult to test very small antigens

Heterogenous Competitive Immunoassays

• Labeled and unlabeled analytes (antigens) compete for a limited number of binding sites on the antibody
• Less label measured in the assay means more unlabeled competitor antigen is present (inverse relationship)
• Concentration of bound label is plotted against the analyte concentration resulting in an inverse standard curve

Homogenous Competitive Immunoassays

• Both the labeled antigen and the unlabeled antigen (from the sample) compete for a limited amount of antibody
• Fewer labeled complexes found when analyte concentrations are higher

Competitive Immunoassays

• Capture antibody is immobilized on a solid phase
• Analyte (antigen) and enzyme-labelled analyte compete for limited antibody binding sites
• Washing removes unbound material
• Enzyme substrate is added to produce a signal

Activity - Comparison of ELISA Procedures

• Sandwich ELISA: Antibody-coated well → analyte addition → enzyme-conjugated antibody addition
• Competitive ELISA: Antibody-coated well → analyte and labelled analyte addition → enzyme-conjugated antibody addition (signals inversely correlate with analyte concentration)

Conjugation & Detection Techniques

• Conjugation is linking either antigen or antibody to another molecule for detection or quantitation
• Antigens or antibodies can be conjugated to various molecules including:
  • Enzymes (e.g., alkaline phosphatase, horseradish peroxidase)
  • Fluorescent labels (e.g., fluorescein isothiocyanate)
  • Biotin/avidin
  • Beads (e.g., polystyrene, magnetic)
  • Isotopic labels (e.g., ³H, ¹²⁵I, ¹⁴C)

This quiz focuses on the principles and design of immunoassays, including the roles of antibodies and antigens in detecting and quantifying compounds. Learn about the characteristics of different assay types, conjugation techniques, and the importance of affinity and specificity in antibody selection.