Electrochemical detection of Toxocara canis excretory-secretory antigens in children from rural communities in Esmeraldas Province, Ecuador: association between active infection and high eosinophilia

Abstract

Background: The diagnosis of active Toxocara canis infections in humans is challenging. Larval stages of T. canis do not replicate in human tissues and disease may result from infection with a single T. canis larva. Recently, we developed a nanobody-based electrochemical magnetosensor assay with superior sensitivity to detect T. canis excretorysecretory (TES) antigens. Here, we evaluate the performance of the assay in children from an Ecuadorian birth cohort that followed children to five years of age. Methods: Samples were selected based on the presence of peripheral blood eosinophilia and relative eosinophil counts. The samples were analyzed by the nanobody-based electrochemical magnetosensor assay, which utilizes a bivalent biotinylated nanobody as capturing agent on the surface of streptavidin pre-coated paramagnetic beads. Detection was performed by a different nanobody chemically labelled with horseradish peroxidase. Results: Of 87 samples tested, 33 (38%) scored positive for TES antigen recognition by the electrochemical magnetosensor assay. The average concentration of TES antigen in serum was 2.1 ng/ml (SD = 1.1). The positive result in the electrochemical assay was associated with eosinophilia > 19% (P = 0.001). Parasitological data were available for 57 samples. There was no significant association between positivity by the electrochemical assay and the presence of other soil-transmitted helminth infections. Conclusions: Our nanobody-based electrochemical assay provides highly sensitive quantification of TES antigens in serum and has potential as a valuable tool for the diagnosis of active human toxocariasis.

Nanobody-Based Immunosensor Detection Enhanced by Photocatalytic-Electrochemical Redox Cycling

“…Taking into account the decrease in sensitivity, the sample dilution (4×) in the ICD procedure, and possible additional sample dilution due to limited sample volume, a margin of sensitivity by a factor of 100 is expected to ensure the detection of TES in real samples, which corresponds to our previous study on detection of TES using HRP-labeled Nbs, leading to an LOD in serum after IDC of 0.9 ng/mL . In the present work, the sensitivity has been improved by a factor of 10, allowing detection at 20–100 pg/mL depending on the dilution factor.…”

“…Production and purification of TES antigens were carried out as previously reported . The concentration of TES in the stock solution was determined from absorbance at 280 nm (OD280 of 1 corresponds to 1 mg/mL) in line with our previous works. − Library construction, selection, production, and purification of Nbs were done according to Conrath et al and described in detail previously . Capture Nb (Nb 2TCE49) was prepared as bivalent-monospecific construct in vivo biotinylated by co-expressing the pACYC184 plasmid (Avidity, USA) containing the pBirACm enzyme.…”

“…The detection format used in the present work (Figure B) was adopted from our previous work on electrochemical detection of TES using Streptavidin precoated magnetic beads and the same Nbs. − However, in the present work, Nb-ALP was used instead of Nb-HRP as detection Nb. In brief, Streptavidin precoated magnetic beads (Dynabeads M-280, ThermoFisher Scientific) were incubated with capture Nb 2TCE49 (2 μg per 1 mg beads) in PBS containing 0.05% Tween 20 (PBST20 buffer) for 1 h on a rotary shaker.…”

“…We have recently developed an electrochemical method based on Nbs, HRP label, and magnetic beads for diagnosis of the active form of human toxocariasis (HT), a parasitic disease caused by the accidental ingestion of Toxocara canis eggs. , Although this is the most sensitive technique to date, the found concentrations of specific antigens in real samples are scattered close to the LOD, warranting further improvement of the method aimed at lowering the LOD.…”

Application of Single-Domain Antibodies (“Nanobodies”) to Laboratory Diagnosis

“…Similar advantages in sensitivity were seen with a growth hormone assay utilizing a nanobody, wherein a detection range of 0.5–110 ng/mL was achieved [ 48 ]. Our efforts to design better performing assays culminated in a recent highly reproducible sensitive and specific assay for Toxocara excretory secretory components [ 49 , 50 ].…”