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.

Fig 1. 2-D gel electrophoresis, silver staining and western blotting of Toxocara canis Excretory Secretory Antigens (TES-Ag).

The TES-Ag sample was separated and analyzed using 2D gel electrophoresis and western blotting. Three of the 2DE gels were transferred to nitrocellulose membranes and probed with a strong EIA positive Toxocara human sera pool (A), a negative human serum sample (B), and Baylisascaris procyonis positive serum (C).A reference gel was stained using silver stain (D). The circled spots in D represent proteins that were excised and subjected to mass spectrometry analysis.

Fig 2. Purity and antigenicity of purified recombinant proteins.

SDS-PAGE of the three recombinant proteins. A. Tc-CTL-1; B. Tc-TES-26; C. Tc-MUC-3. The recombinant protein samples at a concentration of: 1–6.25 ng/mm; 2–3.125 ng/mm; 3–1.6 ng/mm, and 4–0.8 ng/mm was treated with SDS and heated at 65°C for 15 minutes, separated and analyzed using SDS gel electrophoresis and western blotting. Two gels were transferred to nitrocellulose membranes and probed with a strong EIA positive Toxocara human sera pool, a negative human serum sample diluted 1:100 in PBS/Tween 0.3%/5% milk, and one gel was incubated with protein staining, Colloidal Gold Total Protein Stain (Bio-Rad, Cat. # 170–6527). M = Precision Plus Protein Dual Xtra Standards (Biorad, Cat. #161–0377)

Fig 3. ROC Curves of Tc-CTL-1 and Tc-TES-26, and optimal linear combination for visceral larval migrans.

ROC Curves of Tc-CTL-1, Tc-TES-26, and the optimal linear combination were constructed based on Luminex-derived mean fluorescence intensity antigen for visceral larval migrans from 288 negative U.S. serum samples + 134 heterologous parasitic infected serum samples and 204 visceral larval migrans positive serum samples; thresholds are calculated with the highest sum of sensitivity and specificity and are represented on the plot with black dots and corresponding 95% confidence intervals.