Optimizing Conditions for the Yield of One Side Bound IgG Protein Compounds
An antibody, also known as an immunoglobulin, is a large protein that can bind to foreign bodies, e.g. microbes or infected cells, and trigger an attack by other components of the immune system, or can neutralize its target directly. Antibodies are “Y” shaped, with two heavy chains composing the base of the “Y” and a light chain and variable region composing the two upper arms of the “Y”. Research has shown that when attaching molecules to antibodies for use in targeted drug therapies, it is preferable to control the site of attachment so as not to interfere with the antibody’s ability to bind its target antigen. Due to the symmetric nature of antibodies, even when using site-specific conjugation methods, the attached molecule would naturally bind to both sides of the antibody. The presence of molecules on both heavy chains of the antibody, even if not in the antigen binding site, can interfere with normal antibody function. For example, different regions of the antibody are responsible for the long circulation times of antibodies in the blood and other regions are for recruiting immune cells. The hypothesis is that if one side remains unaltered, the impact of the molecule on normal antibody function will be minimal. We developed and compared two methods to label only one of the two heavy chains. Polyacrylamide gel electrophoresis was used to assess the extent of heavy chain modification and to identify the approach that maximized the amount of antibody with only a single modification. This information will aid in further development of antibody targeted drug therapies.