Serological endpoints: Agglutination - Stage 1

Agglutination is the key end-point that we use in the vast majority of TML testing.

Agglutination occurs in two stages:

Stage 1: Sensitization: This involves the initial binding of antibodies to the red cell antigen through non-covalent bonds. The binding of a single antibody to a single red cell antigen is not visible to the naked eye. Several factors can affect the equilibrium constant of the antibody that dictates how strongly it binds to the corresponding antigen. These factors can also be manipulated to remove an antibody from an antigen (e.g. elution).

  • pH – a pH of 6.5 to 7.5 is ideal for most antigen-antibody reactions. Some antibodies react stronger at more acidic pH.
  • Temperature – IgM isotypes react optimally at colder temperatures (e.g. room temperature), whereas IgG isotypes react optimally at warmer temperatures (e.g. 37 C). This is because the IgM antibody-carbohydrate antigen complex is held together through hydrogen bonds: hydrogen bonds are exothermic and have higher stability at colder temperatures. Notably, most IgM antibodies are directed against carbohydrate epitopes; this because carbohydrates, unlike peptides, are ineffective at eliciting a T-helper cell response required for IgG class switching.
  • Ionic strength – A reduction in the ionic strength of the testing medium can increase the rate of association between red cell antigens and antibodies. Low ionic saline solution (LISS) can be used to take advantage of this property.
  • Antibody-antigen ratios – There should be an optimal proportion of antibody and antigen to ensure equilibrium between the rates of antibody association and dissociation. Antibody or antigen excess can disturb this equilibrium. Although the concentration of the red cells may not differ, the zygosity of the antigen may result in higher or lower quantities of the antigen on the red cell surface.

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