Immunologic Laboratory Techniques

Ag-Ab Interactions & Assays - Binding Basics

• Affinity: Strength of single Fab-epitope bond. $K_a = \frac{[AgAb]}{[Ag][Ab]}$.
• Avidity: Overall binding strength of multivalent Ab to multivalent Ag (e.g., IgM: high avidity). Functionally more crucial.
• Cross-reactivity: Antibody binds unintended Ag sharing similar epitope.
• Factors: Optimal pH, temperature (e.g., 37°C), ionic strength. 📌 Mnemonic: PIT (pH, Ionic strength, Temperature).

⭐ High avidity often compensates for low affinity, crucial for diagnostics & in vivo neutralization.

Labeled Immunoassays - Tag & Track Titans

• Principle: Detect Ag/Ab by tagging one component with a measurable label (enzyme, radioisotope, fluorochrome). ↑ Sensitivity.

• 📌 Mnemonic: "RIpE" (RIA, Immunofluorescence, ELISA).

• Types:

  • ELISA (Enzyme-Linked Immunosorbent Assay):
    • Enzyme (HRP, AP) + Substrate → Color.
    • Types: Direct, Indirect, Sandwich (Ag detection), Competitive.
    • Uses: HIV screening, hormones, Abs.

    ⭐ ELISA is a primary screening test for HIV antibodies.

  • RIA (Radioimmunoassay):
    • Label: Radioisotope (e.g., $^{125}$I). Measures radioactivity.
    • Highly sensitive.
    • Uses: Hormones (T3, T4, insulin), drugs.
    • ⚠️ Radioactive material handling.
  • Immunofluorescence (IF):
    • Label: Fluorochrome (FITC, rhodamine). Fluorescence microscopy.
    • Direct (DIF): Ag in tissue.
    • Indirect (IIF): Ab in serum (e.g., ANA).
    • Uses: Autoimmune dx, tissue Ags.

Flow Cytometry - Cell Sorting Celebs

• Analyzes physical and chemical characteristics of single cells or particles as they flow in a fluid stream through a laser beam.
• Key Components: Fluidics system, laser(s), optical detectors, and a computer system.
• Parameters Measured:
  • Forward Scatter (FSC): Proportional to cell size.
  • Side Scatter (SSC): Proportional to cell granularity or internal complexity.
  • Fluorescence Intensity: Detects specific molecules using fluorochrome-labeled antibodies (e.g., CD markers).
• Applications:
  • Immunophenotyping: Identifying and quantifying cell populations (e.g., leukemia/lymphoma classification).
  • CD4+ T-cell enumeration (HIV monitoring; AIDS diagnosis often involves CD4 count < 200 cells/µL).
  • Fluorescence-Activated Cell Sorting (FACS): Physically separates cells of interest based on their properties.

⭐ Gating is a critical step in flow cytometry analysis, allowing for the isolation and examination of specific cell subpopulations from a heterogeneous sample based on their scatter and fluorescence properties.

Western Blotting - Protein Detectives

Highly specific technique to detect target proteins from a complex mixture. Proteins are separated by size via electrophoresis, transferred (blotted) onto a solid membrane, and then identified using specific antibodies.

• Principle: Antigen-Antibody interaction.
• Steps:

• Membranes: Nitrocellulose, PVDF.
• 📌 Mnemonic: SNoW DRoP (Southern-DNA, Northern-RNA, Western-Protein).

⭐ Western Blot is the confirmatory test for HIV, detecting specific viral proteins like p24, gp41, gp120/160.

High‑Yield Points - ⚡ Biggest Takeaways

• ELISA: Detects antigens or antibodies via enzyme-linked reactions; high sensitivity.
• Immunofluorescence (IF): Uses fluorochrome-labeled antibodies; Direct IF for tissue antigens, Indirect IF for serum antibodies.
• Flow Cytometry: Analyzes cell populations (e.g., CD4/CD8 counts) using light scatter and fluorescence.
• Western Blot: Confirmatory for HIV; detects specific proteins separated by electrophoresis.
• Agglutination tests: Visible clumping of particulate antigens with antibodies (e.g., blood grouping).
• Precipitation tests: Soluble antigen-antibody complexes form insoluble precipitates (e.g., VDRL).