The IAT can be used to detect and identify antibodies. The test uses antihuman globulin (AHG) reagent to detect in vitro sensitization of red cells. Patient serum or plasma is incubated with reagent red cells with known antigen phenotypes. If an antibody is present in the serum, it will bind to the reagent red cells with the target antigen, this is referred to as sensitization. A critical step in detection involves initial sensitization, followed by crosslinking to create a reaction visibly observable on the macroscopic level referred to as agglutination. Because most IgG molecules are incapable of producing macroscopic agglutination, AHG is needed to act as a bridge.
The AHG used in the IAT is anti-IgG. Anti-IgG, a manufactured reagent, is most commonly derived from mice that have been stimulated to produce antibodies targeting the Fc region of human IgG proteins (depicted in blue in the image below). Anti-IgG will bind to the patient's IgG antibody if present and facilitate macroscopic agglutination. IgG antibodies present in the patient's serum or plasma are most often considered clinically significant.
One of three common methods used to detect and identify antibodies include:
