Project Lead(s): Cedric Yansouni
Visceral leishmaniasis (VL) causes more deaths per year than any parasitic disease except malaria.
Accurate, non-invasive diagnostic tests that reflect disease activity are urgently needed for the disease.
The project team wanted to develop a unique, low-cost, non-invasive test using an electricity-free platform for visceral leishmaniasis.
The project was based on the premise that monoclonal antibodies against the A2 antigen of Leishmania donovani could be used to develop a lateral flow antigen-detection immunoassay for VL.
The project team had already established that the A2 antigen was a promising target for VL diagnostics development because it is:
· expressed at high levels in the agents of VL (L. donovani and L. chagasi/infantum), but not in other Leishmania species (L. major, L. tropica, L. aethiopica or L. braziliensis)
· multi-epitopic, with 50–100 repeats of the epitope in each protein, and
· detectable by immunofluorescence in confocal microscopy studies of L. donovani as well as L. major transfected with the gene encoding the A2 protein.
Using immunofluorescence with a monoclonal mouse antibody against A2 (mAb-A2), the researchers were consistently able to specifically detect A2 in L. donovani promastigotes in cell culture.
They sought to use mAb-A2 in an Enzyme Linked Immunosorbent Assay (ELISA) format to detect the A2 antigen in whole blood of L. donovani-infected mice, but results were disappointing and were not distinguishable from the negative control blood from uninfected mice.
Despite several intrinsic features that suggest high suitability for a diagnostic assay, the mAb-A1 antibody is not a suitable reagent for detection of L. donovani amastigotes living within infected macrophages of humans suffering from VL.
The antibody is highly sensitive and specific for the detection of L. donovani promastiotes cultured in vitro, but not in axenically cultured amastigotes, suggesting that differential levels of A2 expression in amastigotes, rather than penetration of the mAb-A2 into the intracellular compartments, is a potential explanation for these results.
Limitations of the A2 antigen target means that no functional enzyme immunoassay for A2 that performs with realistic samples from infected mice and humans could be generated.
As a result, subsequent phases of the planned work were not executed.