Characterization of binding reactivity of monoclonal antibodies with α1-antitrypsin conformations to assess their potentials in clinical diagnostic utilities

Abstract

α1-Antitrypsin functions as protease inhibitor and has a key role in regulating neutrophil elastase destructive activity. Misfolding and polymerization of this protein due to genetic attributes, most commonly Z mutation, lead to α1-Antitrypsin deficiency that can progress into liver cirrhosis, pulmonary emphysema and other inflammatory conditions such as panniculitis and vasculitis. This disease can be highly under- recognized thus, more affordable and accessible diagnostic tools are needed to be introduced to the pharmaceutical market. The aim of the project revolves around characterising the binding reactivity 4B12 and 8A7 monoclonal antibodies that might show potential utilities in clinical diagnostic screening. This was investigated through three objectives: firstly, monitoring the polymerization process of fluorescently tagged plasma α1-antitrypsin at real-time showed that 8A7 mAb has more broader polymer specificity as it detected physiological polymers. Secondly, epitope mapping of 8A7 was done using single cysteine plasma α1antitrypsin that is fluorophore-conjugated and induced into polymers using heat. This experiment concluded that 8A7 mAb epitope doesn’t overlap with the other antibodies introduced to the competition assay. Many crystallography trials have been done to obtain AAT:4B12 fab complex crystal protein structure, starting with expressing and expanding native AAT using E-Coli system then purifying the protein to be combined with digested 4B12 fab to co-crystalize them as AAT:4B12 fab complex. Native and cleaved antitrypsin were included in the crystallization trails along with I340W α1-antitrypsin variant. However, more attempts with minor optimization should be done to produce positive crystallography outcome.