Q. Can anti-human globulin (AHG) bind to human antibody without that antibody being bound to an antigen? For example, in forward typing and reverse typing, if you added AHG to anti-A antibodies or anti-B antibodies (free form), would a complex be formed? I’m just confused because AHG is classified as a type of “antibody” but it is not clear to me how it binds, and whether it binds to the antigen, the antibody, or the antigen-antibody complex.

A. Anti-human globulin (AHG) really is just an antibody, just like any other antibody. It is “created” by injecting an animal (mouse, goat, rabbit) with human serum (containing human Ig). The animal will make antibodies to the human Ig, and you can collect those anti-human antibodies and use them in tests. AHG binds to anti-A and anti-B antibodies directly, most likely to their Fc fragments. In the image above, you can see how the AHG binds to the Fc fragments of the antibodies attached to the red cells. AHG will bind to human antibodies whether they are free-form, or whether they are attached to something (like a red cell).

The DAT (and IAT) are cool because the anti-A and anti-B antibodies are bound to red cells. In the DAT, the antibodies are already bound to the red cells in vivo, whereas in the IAT, you mix the antibodies and red cells together in a test tube before adding the AHG. Either way, when you add the AHG, it kind of links the antibodies – and therefore the red cells to which they are attached – together, forming a visual clump! You wouldn’t see any clumping if you combined AHG and free-floating anti-A or anti-B antibodies. They would clump together and form complexes, but because the anti-A or anti-B antibodies were not attached to red cells, there would be no way to detect the complexes.

Other tests work on this same principle of AHG binding to human antibodies (not just anti-A or anti-B, but any old antibodies). Western blot involves the use of enzyme- or radioactive isotope-tagged AHG. You add the tagged AHG to a membrane which has human antibodies attached to it, and then when you add substrate (in the case of enzyme-tagged AHG) or use radioactive detection methods (in the case of radioactive isotope-tagged AHG), you can see where the AHG bound on the strip. ELISA uses AHG too. It’s a cool concept – an antibody that binds to any human antibody. You just have to have a way of detecting that binding. In the case of the DAT and IAT, the antibodies are nicely bound to a big red cell, so you have an easy detection method built right in.