Development of a nanobody-based amperometric immunocapturing assay for sensitive and specific detection of Toxocara canis excretory-secretory antigen

Introduction Human Toxocariasis (HT) is a zoonosis that,
despite of its wide distribution around the world, remains poorly
diagnosed. The identification of specific IgG immunoglobulins
against the Toxocara canis Excretory-Secretory antigen (TES), a
mix of glycoproteins that the parasite releases during its
migration to the target organs in infected patients, is currently
the only laboratory tool to detect the disease. The main
drawbacks of this test are the inability to distinguish past and
active infections together with lack of specificity. These factors
seriously hamper the diagnosis, follow-up and control of the
disease.
Aim To develop an amperometric immunocapturing diagnostic
assay based on single domain immunoglobulins from camelids
(nanobodies) for specific and sensitive detection of TES.
Methods After immunization of an alpaca (Vicugna pacos)
with TES, RNA from peripheral blood lymphocytes was used as
template for cDNA amplification with oligo dT primers and
library construction. Isolation and screening of TES-specific
nanobodies were carried out by biopanning and the resulting
nanobodies were expressed in Escherichia coli. Two-epitopes
amperometric immunocapturing assay was designed using
paramagnetic beads coated with streptavidin and bivalent
nanobodies. Detection of the system was carried out with
nanobodies chemically coupled to horseradish peroxidase. The
reaction was measured by amperometry and the limit of
detection (LOD) was compared to conventional sandwich
ELISA.
Results We obtained three nanobodies that specifically
recognize TES with no-cross reactivity to antigens of Ascaris
lumbricoides and A. suum. The LOD of the assay using PBST20
0.05% as diluent was 100 pg/ml, 10 times more sensitive than
sandwich ELISA.
Conclusion Sensitive and specific detection of TES for
discrimination of active and past infections is one of the most
difficult challenges of T. canis diagnosis. The main advantage of
our system is the use of two different nanobodies that
specifically recognize two different epitopes in TES with a highly
sensitive and straightforward readout. Considering that the
amounts of TES available for detection in clinical samples are in
the range of picograms or a few nanograms maximum, the LOD
found in our experiments suggests that the test is potentially
useful for the detection of clinically relevant cases of HT.