Tuberculosis has re-emerged as a global health problem due to co-infection with HIV and the emergence of drug resistant strains of Mycobacterium tuberculosis. There is a need for a reliable and fast serodiagnostic assay to reduce the time required for test results from weeks to hours, in order to better control the spread of the disease. Previous studies have shown that TB patients contain antibodies against M. tuberculosis mycolic acids. In standard immunoassays such as ELISA, an unacceptable number of false positive and negative test results were obtained. This study aimed at assessing the potential of detecting anti-mycolic acids antibodies in TB patient sera on a biosensor as surrogate marker for TB infection. Mycolic acid liposomes were immobilized reproducibly on a non-derivatized biosensor cuvette and blocked with saponin. A high dilution of serum in PBS/ AE was used to calibrate the signal of the two cells, followed by binding of patient sera inhibited with either mycolic acid, cholesterol or placebo phosphatidylcholine liposomes at a lesser dilution. The inhibition was done to confirm the specificity of the binding response. There was no inhibition of binding when a sputum negative control serum (HIV-TB-) was pre-incubated with either cholesterol or mycolic acids on the biosensor coated with mycolic acid liposomes. The antibodies that are specific to mycolic acid were demonstrated in all TB positive patients on mycolic acids coated cuvette cell surfaces after pre-incubation of serum with mycolic acids. The patient sera that were false positive and false negative on ELISA tested negative and positive respectively on the biosensor. Only sera from two patients, both HIV positive, tested false positive on both ELISA and biosensor. The biosensor was able to detect anti-mycolic acids antibodies of even low affinity. In ELISA, these antibodies were washed away. No inhibition of antibody binding on cholesterol-coated cuvettes was found after pre-incubation of serum with mycolic acids or cholesterol liposomes. The cholesterol surface became unstable during pre-incubation of serum with mycolic acids. Mycolic acid appeared to be a stronger antigen than cholesterol. The anti-mycolic acids antibodies were specific and sensitive for diagnosis of TB on the biosensor. More sera should be analyzed on the biosensor to make a statistically accountable statement on whether the improved sensitivity and specificity is adequate for a simple, rapid, sensitive and accurate biosensor-based serodiagnostic assay.
Dissertation (MSc(Biochemistry))--University of Pretoria, 2006.