Strongyloides-Specific IgE Phage cDNA Clones and Development of a Novel ELISA for Strongyloidiasis

Abstract

Strongyloidiasis, caused mainly by the nematode Strongyloides stercoralis, is prevalent worldwide and potentially fatal in immunosuppressed patients. We report on a new IgE biomarker to diagnose Strongyloides infection. Sera from two groups infected with Strongyloides served as positive samples: Group 1A, in which infection was confirmed by stool-microscopy and/or stool-polymerase chain reaction (PCR) and was seropositive by an IgG-enzyme linked immunosorbent assay (ELISA) and an IgG4 rapid test, and Group 1B in which infection was confirmed by stool-PCR but was seronegative. Negative samples (controls) comprised infections with other parasites (Group II) and healthy donors (Group III). Immunoscreenings of an S. stercoralis complementary DNA (cDNA) library were performed, and the cDNA clone with the highest diagnostic potential (clone A133) was selected for recombinant protein production and then evaluated using IgE Western blot and ELISA. The Western blot showed that the recombinant protein (rA133) was 100% reactive with Group IA (n = 10) and Group IB (n = 5), and 96% non-reactive with Groups II and III (n = 25). Subsequently, the IgE-ELISA was developed and showed 100% diagnostic sensitivity in Groups IA (n = 32) and IB (n = 11); and 99.3% specificity in Groups II and III (n = 144). In conclusion, this study has identified rA133 as a novel recombinant protein with potential diagnostic value, and that the IgE-ELISA incorporating this protein may be useful for patient diagnosis and epidemiological studies.

Keywords: IgE-enzyme linked immunosorbent assay (ELISA); Strongyloides stercoralis; complementary DNA (cDNA) library; immunoscreening; recombinant antigen; serodiagnosis; strongyloidiasis.

Figure 1

(A) SDS-PAGE analysis of 1, 5, and 10 µg of pooled rA133 from the eluted fractions after purification. The ~40 kDa protein band corresponds to rA133; (B) Western blot analysis of 1, 5, and 10 µg of purified His-tagged rA133 using Anti-His HRP as a probe. The 40 kDa protein band corresponds to r A133; (C) IgE Western blot analysis of rA133. The arrows show the rA133 protein bands on the nitrocelulose strips. Strip M is the unstained protein marker (Bio-Rad). Positive samples (strongyloidiasis sera) were tested as follows: Group IA serum (strips 1–2) and Group IB (strips 3–4). Group II control (other infections) serum samples were tested as follows: ascariasis (strips 5–7); filariasis (strips 8–10); hookworm infection (strips 11–13); toxocariasis (strips 14–16); schistosomiasis (strips 17–18); giardiasis (lane 19). Group III healthy individuals were tested with strips 20–22. A 40 kDa protein band that corresponded to rA133 was observed in strips 1–4. Strip 13 tested with serum from hookworm infection showed a false-positive result.

Figure 2

Receiver operating characteristic (ROC) analysis of the IgE-ELISA using rA133. The cut-off value (COV) is 0.22 and the area under the ROC curve (AUC) is 0.9998 (95% confidence interval = 0.9993 to 1.000).

Figure 3

One-way ANOVA with Bonferroni multiple comparison test of the optical density (OD405) values (mean + 1SD) of all serum groups. All the groups have significant p-value < 0.0001 except for Groups II and III, which have statistically nonsignificant p-value > 0.05.

Figure 4

The distribution of optical density (OD₄₀₅) values of different serum groups by IgE-ELISA. The cut-off value OD₄₀₅ value (COV) to discriminate between positive and negative results was 0.22.