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Blood Bank

Ag-Ab Interactions

For immunohematology testing, we leverage the specific, yet reversible, binding properties of antibodies and antigens. Because many clinically significant interactions (especially IgG) don’t cause direct agglutination, we rely heavily on the Antiglobulin Test (or newer sensitive methods like Solid Phase/Gel) to detect this crucial sensitization step, ensuring safe transfusions

The Fundamental Principle: Specificity (The “Lock & Key”)

  • Antibodies are designed to recognize and bind to one specific epitope (a small part of an antigen) using their unique antigen-binding site (paratope) located on the Fab arms
  • This binding relies on a precise 3D shape and chemical match
  • Why it matters: This specificity allows us to use known antibodies (reagents) to identify antigens on cells (like in blood typing) or use known antigens (reagent cells) to detect antibodies in plasma (antibody screening/ID)

The Nature of the Bond: Weak Forces & Reversibility

  • Binding isn’t permanent; it’s based on multiple weak non-covalent forces (hydrogen, ionic, van der Waals, hydrophobic)
  • These forces require very close proximity between the antibody and antigen
  • The interaction is reversible (Ag + Ab <=> AgAb), following the Law of Mass Action – concentrations of Ag and Ab drive the reaction
  • Why it matters: Factors influencing proximity (like ionic strength or cell repulsion/zeta potential) affect binding, especially for IgG. Reversibility necessitates careful washing steps in some tests

Binding Strength: Affinity vs. Avidity

  • Affinity: Strength of a single binding site to a single epitope
  • Avidity: Overall strength of binding when multiple sites are involved (like pentameric IgM binding to multiple antigens on a cell). High avidity can compensate for lower affinity
  • Why it matters: High avidity (IgM) often leads to direct agglutination. IgG relies more on affinity and often needs help (AHG) to be detected

Making it Visible: Testing Principles & Methods

  • Since binding itself is invisible, we need methods to detect it, usually by looking for agglutination (clumping) or hemolysis (cell lysis)
  • Two Stages
    1. Sensitization Antibody binds to antigen on the red cell (invisible). Influenced by temp, time, pH, ionic strength
    2. Lattice Formation Antibody cross-links multiple cells -> visible agglutination. Influenced by antibody class (IgM >> IgG), cell distance, centrifugation

Key Testing Methods

  • Direct Agglutination (e.g., Tube - Immediate Spin/RT): Relies on antibodies (mainly IgM) being large enough to bridge the gap between cells in saline. Used for: ABO typing, detecting strong cold IgM antibodies
  • Antiglobulin Test (Coombs Test): Detects “invisible” sensitization (usually IgG or Complement) using Anti-Human Globulin (AHG) reagent as a bridge
    • Direct Antiglobulin Test (DAT): Detects if patient RBCs are coated in vivo (in the body). Used for: HDFN, AIHA, HTR investigations
    • Indirect Antiglobulin Test (IAT): Detects antibodies in patient plasma capable of coating reagent RBCs in vitro (in the lab). Used for: Antibody screening/ID, crossmatching, weak D testing. Requires incubation (often with enhancement media like LISS or PEG) and thorough washing before adding AHG. Check cells validate negative results
  • Solid Phase Red Cell Adherence (SPRCA): Immobilizes antigen or antibody on microplate wells; positive reaction = adherence of indicator cells. Standardized, sensitive
  • Gel Technology (Column Agglutination): Uses microtubes with gel; agglutinated cells get trapped during centrifugation. Standardized, clear endpoints, widely used