FIND and SBRI collaborate in selection of diagnostic reagents for
trypanosome antigens
Among the most commonly used tools for the
diagnosis of sleeping sickness is a serological test for exposure to the
parasite Trypanosoma brucei gambiense. A limitation of such tests is their
inability to distinguish active infections from past or subclinical exposures. A
direct test for parasite proteins in blood samples would offer better
specificity for active infection. Unfortunately, past attempts to develop such
assays have not been successful, mainly due to inadequate sensitivity. New
molecular sensing methods developed in recent years could offer better
sensitivity for detection of parasite protein. Many such methods are inexpensive
and easy to use at point of care, even in remote locations such as the ones
where sleeping sickness occurs. With these innovations, detection of pathogen
protein has become a more attractive option for diagnosis of infectious
diseases.
However, a critical bottleneck remains: whereas diagnostic platforms
are extensively optimized, the same is not always true of the detection reagents
that go into them. Binding reagents that specifically recognize target molecules
in patient samples constitute the core of any "state of the art" diagnostic
test. When proteins are being detected, the most commonly used probes are
antibodies, the proteins produced by mammals that confer pathogen-specific
protection against disease. Very often, the sensitivity of a diagnostic test is
limited by an inadequate affinity of antibodies for their molecular targets.
Advantages of antibody
probes in cancer
imaging and sleeping
sickness diagnosis
New methods enable researchers to generate
and optimize antibody probes in the laboratory.
Ideal probes have high affinity for
the target protein with no cross-reactivity,
and are stable at working temperature.
Engineered antibody fragments, such as
single chain variable fragments (scFv), have
additional advantages in terms of manufacturing
economy. Moreover, because they are
much smaller in size than naturally-occurring
mammalian antibodies such as IgG, they can
penetrate to molecular targets that cannot
be detected by traditional antibody probes.
For these reasons, engineered antibody
probes have become modern tools in cancer
imaging and diagnosis.
FIND is working with the Seattle Biomedical
Research Institute (SBRI) to apply scFv antibody
engineering technology in the development
of optimized antibody probes for
trypanosome proteins in blood. Using a technology
called yeast display, high-affinity
probes for a number of T. brucei proteins will
be generated, and those which are best suited
for diagnostic detection in human samples will
be identified. Finally, the sensitivity and
stability of the probes for the chosen proteins
will be further enhanced by antibody engineering
methods. The outcome will be a set
of antibody probes with characteristics of
sensitivity, stability, and manufacturability
that are superior to probes generated by traditional
methods. This general approach has
potentially broad applications in immunological
test development beyond human African
trypanosomiasis diagnostics. It will also facilitate
the validation of novel biomarkers, a
critical bottleneck in biomarker discovery.
Figure: Affinity maturation of scFv antibodies. Panel A
shows a parental clone selected for binding to the recombinant
HIV Ag gp140 at 50 nM, and affinity stained with 50 nM
biotinylated Ag. Panels B through D show sequential selections
conducted on the mutagenic library generated from this clone
using Ag at 5 nM. The black box in the upper right quadrant
was used as a sort gate, and the sorted cells became the population
of each successive selection. Clones in the upper left quadrants
were specific Ag-binding clones that stained poorly with
the scFv expression stain, anti-c-myc, due to mutagenesis.
The same approach is being used by SBRI for HAT.
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