The effect of point mutations on the binding affinity of anti-blood group A antibody AC1001.

Abstract

The A and B blood group antigens are very similar, and differ in only one functional chemical moiety; nevertheless this single difference is enough to allow for the recognition of these carbohydrate antigens by their corresponding anti-blood group antibodies. This thesis is focussed on an anti-blood group A (BGA) antibody interaction with its A antigen. A single-chain variable-domain antigen binding fragment (scFv) gene was constructed based on the known sequence of an anti-BGA monoclonal antibody (AC1001), and the protein has been expressed in E. coli. The objective of this work is to improve the binding affinity of this BGA scFv for the A-antigen, and to better understand the mechanisms by which the anti-blood group antibodies can discriminate between the largely similar A and B carbohydrate antigens. The anti-BGA antibody produced in vivo in the immune system does not undergo the process of affinity maturation and somatic mutation because it binds a T cell independent carbohydrate antigen. Therefore, while these anti-carbohydrate IgM antibodies are able to bind their specific antigen, they do so with a very weak binding affinity. We have mimicked the process of affinity maturation and somatic mutation in vitro and attempted to produce a BGA scFv with an increased affinity using two different approaches. (Abstract shortened by UMI.)