Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26

Abstract

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.

Prevalence of parasitic infections among recent immigrants to Chicago

“…Antibody responses to cysticercosis, strongyloidiasis, and toxocariasis were determined using a multiplex bead-based assay (MBA). [51–53] For cysticercosis, 3 recombinant antigens (rGP50, rT24H, and sTs18var1) were used; one recombinant antigen was used for both strongyloidiasis (Ss-NIE-1) and toxocariasis (rTc-CTL-1). Briefly, the antigens were coupled to carboxylated magnetic particles, and then incubated with sera (diluted 1:100 in PBS/0.3% Tween-20/5% milk) for 30 minutes.…”

xMAP Technology: Applications in Detection of Pathogens

“…In the context of animal health and human food consumption, a list of the top ten parasites has been defined by the UN's Food and Agriculture Organization (FAO) and World Health Organization (WHO) ( Table 3). Although in the last decades a number of novel diagnostic methodological approaches has been developed, the current diagnosis of some parasitic diseases is still based only on a combination of clinical signs, anamnesis, and direct visual identification of parasitological objects (Anderson et al, 2015). The most common conventional diagnostic methods, such as microscopic examination, biochemical assays or ELISA, are available, but they are laborious, time-consuming and in many cases not reliable (Navidad et al, 2013).…”

“…With the rising popularity of MBMIs, ISA was also developed for other members of Nematodes, such as representatives from the genus Toxocara (Anderson et al, 2015). The infection by these parasites is typically peroral at areas contaminated by embryonated roundworm eggs, e.g., sand from childrens' playgrounds.…”

Habit of cooking pork on hot stones as main risk of cysticercosis

“…The samples were prepared using the Luminex Immunoassay technique (14,15) i.e. the blood sample on filter paper was dissolved in 1 x PBS solution (Sigma, Cat.# P4417-100 tab); 0.3% Tween-20 (Sigma, Cat.# P1379 100 ML) and 0.1% NaNO 3 (HiMedia, Cat.# GRM1038), so that the sample concentration was 1:10.…”