Towards paper-based micro bio-sensing of biomarker anti-mycolic acid antibodies for TB diagnosis

Abstract

Accessible point of care diagnosis of Tuberculosis (TB) is an essential development to better manage the global epidemic that infects 10 million people annually. Current diagnostics are centralised, causing patient loss to follow up or delays in treatment. Although serological diagnosis using finger-prick blood has been historically insensitive in the diagnosis of TB, the detection of anti-mycolic acid (MA) antibodies in patient sera has been shown to allow accurate diagnosis in HIV-positive, previously infected and TB exposed patients. MA is a unique lipid antigen of mycobacteria with anti-MA antibodies being formed early upon infection in a T-cell independent pathway. This work aimed to contribute towards the development of a lateral flow immunoassay termed MALIA (Mycolate Antibodies Lateral flow Immuno Assay). This diagnostic has the unique lipid antigen MA as the immobilised capture agent and custom developed monoclonal anti-MA chicken antibodies (gallibodies) as the labelled bio-recognition element. Casein hydrolysate was newly applied as a blocker in enzyme-immuno assay (EIA) to characterise the functionality of the gallibodies in order to circumvent import restrictions on bovine milk products. MA dissolved in hexane and immobilised on nitrocellulose was not detected by the passively conjugated gold labelled gallibody conjugate in the lateral flow test (LFT) format, despite the confirmation with lipid staining and EIA that MA remained immobilised and antigenic on nitrocellulose. Various substrates, blockers and running buffers were explored for the LFT to attempt to detect MA. The method of passive conjugation of the gallibodies to gold nanoparticles was chosen as this is a commonly successful and simple strategy for conjugate preparation in LFTs. Initial characterisation of gold labelled gallibody conjugate suggested that the orientation of the gallibody on the nanoparticle may be favourable for binding by anti-chicken antibody (the control) but not MA. The biological activity (MA binding) of the gold labelled conjugate was probed on alternative methods. The results showed that in EIA, gold labelling caused the loss of MA binding but not anti-chicken immunoglobulin binding. This is possibly due to the extra force in the wash steps caused by the presence of the gold nanoparticle. Interaction of gold labelled gallibody conjugate with antigenic MA nanoparticles in transmission electron microscopy showed loss of biological activity, while dynamic light scattering intensity measurement of the same interaction showed a weak interaction similar to that seen between gold labelled bovine serum albumin conjugate with fatty acid coated nanoparticles. To address the challenges uncovered by this research, gallibodies can be re-engineered to increase functional affinity (by increasing the valency) to be able to compensate for activity losses due to labelling. In addition, labelling of MA, rather than gallibodies may result in a successful, inverted MALIA to meet the ultimate aim to drastically change the face of the TB epidemic at the critical fault line – point of care diagnosis. The promising avenues uncovered by this key explorative research must be pursued to actualise this critically important and non-standard LFT technology.