Immunologic Laboratory Techniques

Immunologic Laboratory Techniques

Immunologic Laboratory Techniques

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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).

Antigen-antibody binding forces

⭐ 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.

Antibody-Antigen Binding in Immunoassays

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. Flow cytometry dot plot showing cell gating

⭐ 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.

Western Blot Workflow

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).

Practice Questions: Immunologic Laboratory Techniques

Test your understanding with these related questions

All of the following forces are involved in antigen-antibody reactions, except:

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Flashcards: Immunologic Laboratory Techniques

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Vascular rejection in organ graft is a type of type _____ hypersensitivity reaction*mechanism?

TAP TO REVEAL ANSWER

Vascular rejection in organ graft is a type of type _____ hypersensitivity reaction*mechanism?

II

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