Hemagglutination

Hemagglutination is an assay based on the ability of antibodies binding to their antigen to alter the physical state of the bound antigen. In contrast to the ELISA, that works on the principle of directly measuring antibody to antigen binding, hemagglutination detects secondary interactions, as the clumping/agglutination of large particles, red blood cells (hema-) containing antigen on their surface by the respective specific antibodies. Binding of the antibodies to their antigen found on the surface of the red blood cells causes hemagglutination. Hemagglutination is usually utilized to determine the ABO blood group of blood donors and transfusion recipients. Cells agglutinate when crosslinked by antibodies, requiring antibodies to have at least two identical antigen-binding sites. Hemagglutination is similar to the precipitin reaction. Lattice formation between antibody (multivalent) and multivalent antigen containing cells leads to agglutination, with no agglutination in areas of excess antibody or antigen. Agglutination can be caused by the primary, first antibody, in direct agglutination, or by a second antibody directed against the first, in indirect agglutination. Agglutination using antibodies to antigenic components adsorbed to red cells (or inert particles) is called passive hemagglutination (passive agglutination) (1,3,4).

Simple Hemagglutination Experiment

In a simple experiment, TNP-specific conjugated sheep red blood cells can be utilized to test whether antibodies present in the supernatant can mediate hemagglutination. This experiment is performed in both the presence and absence of anti-IgM antibodies, which provide further information on the valency of the IgM molecules. Agglutinated red blood cells will occur if the antibody can crosslink them. Monomeric IgM is incapable of hemagglutination at the level of efficiency that polymeric IgM is capable. Addition of the anti-IgM will aggregate the monomeric IgM, allowing for the agglutination of the red blood cells like a polymeric IgM. You are therefore capable of assessing, which IgM supernatants contain the ability to form pentamers and/or hexamers and which could not. Nonpolymeric IgM will form red "bottoms" on the microtitre, while polymeric IgM will not, but form the agglutinate at the side of the well. The addition/subtraction of anti-IgM would depict IgM that need antibodies to aggregate them into polymerics to form the hemagglutination reaction.

Agglutination Reaction

The agglutination reaction has become less used for research and diagnosis, because it is not possible to precisely quantitate the reaction (with modern technology, such as light diffraction it is possible). Moreover, there is a high percentage of error, as failure to observe the presence or absence of agglutination in one tube or one doubling dilution is representative of a 100% error. Visualization of precipitates can be erroneous. Therefore, agglutination is seldom used, being replaced more often by various types of immunoassays (3).

Anti-Mu Antibody

Addition of anti-mu antibody, an appropriate amount, would be able to aggregate any IgM, which is monomeric, rather than polymeric, enabling for agglutination of the red blood cells. This would allow us to reveal if any secreted IgM, that could not agglutinate or fix complement efficiently like polymeric IgM, to be due to a mutation that affected IgM from polymerizing, rather than due to a V-region mutation, because no agglutination would be visible in monomeric, with added non anti-mu, IgM.

References:

1. Janeway C.A., Travers P., Walport M., and Capra J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA pages 1-40, 2.5-2.22, 3.1-3.12

2. Delves P., and Roitt I. 1999. Encyclopedia of Immunology. Academic Press Inc., 2nd ed., San Diego, USA

3. 1994 Current Protocols in Molecular Biology. Volume 2. John Wiley & Sons Inc., USA

4. Cruse J., and Lewis R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA pages 1960-1965

5. Chen F.H., and Painter R.H. 1997. Domain switched mouse IgM. IgG2b hybrids indicate individual roles for C domains in the regulation of the interactions of IgM complement C1q. J. Immunol. 159, 3354-3363