Nanobody-based sandwich enzyme-linked immunosorbent assay for the detection of Toxocara canis excretory secretory proteins.

Abstract

IntroductionHuman Toxocariasis (HT) is a neglecteddisease resulting from tissue invasion of L2larva fromToxocaracanis. The only laboratory diagnostic tool currently available is aserological test detecting IgG against the excretory secretoryproteins of the parasite (TES). This method is unable todistinguish between current and past infections. We developed asandwich enzyme-linked immunosorbent assay (ELISA) takingadvantage of the inherent features of specific single variabledomain fragments of camelids (nanobodiesâ).Methods and MaterialsAn alpaca was immunized with125lg of TES 5 times in intervals of 7 days. One week after thelast immunization, peripheral blood was extracted andnanobodyâsequences were amplified through reverse-transcription polymerase chain reaction (RT-PCR) from bloodlymphocytes. Selection of binders was performed by biopanningbased on phage display. A sandwich ELISA was set up with acapturing nanobodyâcloned in pHEN6c vector and a detectionnanobodyâin vector pBAD17 containing an AviTagTMforin vivobiotinylation. Cross-reactivity was tested with excretoryantigens ofAscaris lumbricoidesandA. suum. Immunocapturingwith paramagnetic beads was used to isolate the specific fractionof TES recognized in the sandwich ELISA.ResultsNanobodyâsequences were present in 3x108transformants. 84% of them contained a plasmid encoding 20different nanobody sequences. The combination 1TCE39 and1TCE52 had the best Optical Density (OD) signal in sandwichELISA with no cross-reactivity withA. lumbricoidesorA. suum.The detection limit using ELISA sandwich format in negativesamples spiked with TES was 40 ng/ml. Immunocapturingdemonstrated that the epitopes recognised by the sandwichELISA are located in the 120 kDa fraction of TES.ConclusionsTests to diagnose active HT are currently notavailable, hampering the estimation of the real prevalence of thedisease and its control. Nanobodyâ-based sandwich ELISAprovides an innovative approach to detect TES ofT. canisinclinical samples.DisclosureThis project is funded by the ResearchFoundation Flanders (FWO