Antibodies possess a number of functional groups, both the amine and carboxylate groups are plentiful. For this reason, conjugation procedures that utilize these groups will cross link randomly to all parts of the antibody molecule. The distribution of amine and carboxylate groups on a three dimensional structure of an immunoglobin is nearly uniform throughout the surface topology. Conjugation to these functional groups lead to random conjugation and hence, random orientation of the antibody often blocking the antigen binding site. Obscuring the binding site will decrease antigen binding activity in the conjugate. Site directed conjugation generally leads to conjugation chemistry successful at preserving activity of the antibody. Conjugation through sulfhydryl of a fragmented antibody is one such method of site directed conjugation. In this method the conjugation takes place at certain positions on the immunoglobin surface that is far from the antigen binding sites thus, preventing blockage of these sites and preserving activity.
The Fab’ has an indigenous sulfhydryl group which can be used for conjugation to maleimide containing lipids. Enzymatic digests of IgG through pepain and pepsin produces two particularly useful fragments called Fab and F(ab’)2. Pepain and pepsin cleave immunoglobin at the hinge region of the heavy chain pairs. In pepsin digestion the disulfide region in the hinge region remain with the antigen binding fragment producing F(ab’)2. The divalent molecule no longer contains an extended Fc portion differing it from a normal intact antibody. Pepain digestion occurs at a point above the disulfide bond releasing two antigen binding sites of an antibody. This type of antibody digestion forms individual dimeric fragments (Fab) containing one antigen binding site each.
Using reducing reagents such as DTT, TCEP, or MEA the disulfides between the heavy chains can be cleaved releasing two half antibody molecules, each containing one heavy chain, one light chain and an antigen binding site. Antibody reduction is done in presence of EDTA to prevent reoxidation of the sulhydryls by metal catalysis. The reducing reagents such as DTT and TCEP are kept at 3-fold molar excess over the antibody concentration and the reaction is done at a pH of 8.0. Monoclonal antibody reduction may not be completely efficient as some of these antibodies may consist of a high number of disulfides in the hinge region. Conversely polyclonal preparation works quite well with this procedure. Adequate desalting of the reduced antibody from excess reducing agents is recommended. Even a small amount of thiol reductant may interfere with subsequent conjugation with maleimide modified lipids.