Introduction to Immunoanalysis Techniques

Introduction to Immunoanalysis

• Immunoanalysis uses antibody-antigen interactions for detecting and quantifying substances (e.g. hormones, drugs, infectious agents) in biological samples.
• These techniques are vital laboratory tools.

Types of Immunoassays

• Competitive Immunoassays: Analyte competes with a labeled standard to bind to limited antibody. Bound label declines as analyte concentration rises.
• Non-competitive Immunoassays: Analyte binds to antibody; bound label increases with analyte concentration.

Key Techniques in Immunoanalysis

• Enzyme-linked immunosorbent assay (ELISA): A widely used technique using antibody-coated surfaces and various formats (direct, indirect, sandwich, competitive) to quantify substances.
• Radioimmunoassay (RIA): Historically used radioactive labels. Less common due to safety concerns.
• Immunofluorescence: Visualizes and measures antigens using fluorescent-labeled antibodies. Often used in microscopy and cell-based assays.
• Immunoprecipitation: Separates antigens or antibodies using specific antibodies or antigens.
• Immunoblotting (Western blotting): Separates proteins by electrophoresis, then uses antibodies to detect specific proteins.

Principles and Mechanisms

• Specificity: Antibodies bind precisely to specific antigens. This allows for detecting minute quantities in complex samples.
• Sensitivity: These assays can detect very low concentrations (e.g. critical hormones).
• Antibody-Antigen Interaction: Strong and specific binding between antibodies and antigens forms the core of these techniques.

Applications in Diagnostics

• Disease Diagnosis: Essential for identifying and monitoring infectious agents (HIV, bacteria) and autoimmune conditions.
• Hormone Measurement: Crucial for diagnosing and managing endocrine disorders.
• Drug Monitoring: Quantifies drug levels for therapeutic drug monitoring and toxicity assessment.
• Pregnancy Tests: A common example, detecting pregnancy hormones.

Factors Affecting Immunoassay Results

• Sample Preparation: Accurate sample handling is critical for reliable results.
• Reagent Quality: High-quality reagents ensure assay validity.
• Assay Procedure: Following the correct protocol maintains accuracy and reliability.
• Operator Training: Trained personnel minimizes errors and ensures consistent results.

Advantages of Immunoanalysis

• High sensitivity and specificity for target substances.
• Relatively easy implementation using various sample types (serum, plasma, urine, tissue).
• Many commercially available reagents and kits are readily available.

Disadvantages of Immunoanalysis

• Antibody preparation can be costly and challenging.
• Some procedures can be time-consuming.

Further Developments

• Research constantly improves sensitivity, specificity, and speed of immunoassays.
• Automation to increase throughput and reduce errors is ongoing.
• Point-of-care immunoassays are developing for rapid on-site diagnostics.