Abstract:
Tuberculosis (TB) is an infectious disease that is caused by the Mycobacterium tuberculosis
(M.tb) species of bacteria. Despite this being discovered for over 100 years the disease
continues to cause epidemics worldwide. The diagnosis of TB is a challenging aspect, with the
current tests exhibiting many problems including: long time period between testing and
accurate diagnosis, not enough sensitivity, not always accurate, and, in some cases, expensive.
Thus novel biomarkers are urgently needed to aid in the management of the disease. Mycolic
acids (MAs) are complex lipid molecules that are found in the cell wall of mycobacteria and in
particular in M.tb. The fact that these lipids are species-specific makes them a key component
for understanding the M.tb organism. While the isolation of these compounds from the
organism can be a complex and costly process, the production of stereo-controlled, chemically
synthetic tuberculous, mycobacterial MAs can now be conducted in the laboratory. In TB
patient sera, antibody immune activity to MA is always accompanied by antibody immune
activity to cholesterol. Investigations into the cholesteroid nature of MA have been underway
for many years. Researchers have tried to find the MA subclass responsible for this
characteristic of the lipids. In this study, we apply recombinant monoclonal antibodies to MA
to correlate the cholesteroid functionality of mycolic acids to their structure by means of crossreactivity
of antigen recognition. This thesis reports the generation of three different chickenderived
phage-displayed single-chain variable fragments (scFv) that reacted similarly towards
the natural mixture of MA, but differently to the three individual classes. The first antibody
recognized all three classes of chemically synthetic MAs, the second only the two oxygenated
types of MAs and the third only methoxy MA. The cholesterol cross-reactivity was investigated
after grafting each of the three scFv types onto two configurations of constant chain domains
– CH1-4 and CH2-4. Weak but significant cross-reactivity with cholesterol was found only
with CH2-4 versions, probably due to the enhanced binding cooperativity, aiding in the sensitivity. Cholesterol was only recognized by the two monoclonal antibodies that also were
capable of recognizing trans-keto-mycolic acids, suggesting that the cholesteroid nature of
mycolic acids is determined by the conformation of MA that is induced by this stereo-isomer
of keto-mycolic acids. The 2017 World Health Organisation ministerial conference held in
Moscow emphasized the dire need for faster acting drugs and technologies that quickly
diagnose TB and determine the degree of drug resistance to improve the management of TB
worldwide. It is anticipated that the gallibody tools developed here will find application in
quick, affordable TB screening tests aimed at regular monitoring of people at risk of infection
as well as to determine how TB patients respond to their anti-TB therapies.
