Evaluation of a set of refolded recombinant antigens for serodiagnosis of human fascioliasis

Abstract

Diagnosis of fascioliasis with high sensitivity and specificity antigens play a vital role in the management of the disease. Majority of commercial serological tests use F. hepatica native antigens and indicate wide diversities in test accuracy. Nowadays, recombinant antigens have been introduced as diagnostic reagents offer better test standardization. A combination of highly pure recombinant antigens associated with correct folding will leads to improve specificity and sensitivity of ELISA for diagnosis of Fascioliasis. In this article, Fasciola hepatica saposin-like protein 2 (SAP-2), ferritin protein (Ftn-1) and leucine aminopeptidase (LAP) recombinant antigens were considered as tools for the detection of F. hepatica immunoglobulin G antibodies in persons with chronic human fasciolasis. The recombinant antigens were obtained as fusion proteins, expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC). The refolding processes of denatured recombinant proteins were performed using dialysis method in the presence of chemical additives, and reduced/oxidized glutathione (in vitro). The immunoreactivity of the recombinant antigens was assessed individually and in a combination compared with excretory/secretory antigen (E/S) in an enzyme-linked immunosorbent assay (ELISA) test. The experiments were optimized using 213 serum samples from humans, including patients with chronic fascioliasis, patients with other parasitic diseases, and healthy subjects. The results indicated 95% sensitivity and 98% specificity for rtFhSAP-2, 96% sensitivity and 91% specificity for E/S, 80% and 83.3% for rtFhFtn-1, 84% and 88% for FhLAP, and also, 96% and 95% for combination of recombinant antigens, respectively. In conclusion, the results of this investigation showed that rtFhSAP-2 with the highest specificity and acceptable sensitivity has a considerable superiority compared to mentioned antigens and even in combination with these antigens in serodiagnosis of human fascioliasis.

Fig 1. Optimization of recombinant proteins expression in Rosetta (DE3) strain.

The expression of (A) rtFhSAP-2 (B) rtFhFtn-1 and (C) rFhLAP. Bacterial samples which were taken just before (BI) and after induction (AI) with IPTG, examined on 12% SDS-PAGE.

Fig 2. Purification of soluble form of rtFhSAP-2, rtFhFtn-1 and rFhLAP proteins.

(A) Purified (eluted) rtFhSAP-2 after IMAC (1). (B) Purified (eluted) rtFhFtn-1 after IMAC (2). (C) Purified (eluted) rFhLAP after IMAC (3).

Fig 3. Western blot analysis of refolded proteins.

Reactivity of rtFhSAP-2 (1), rFhLAP (2) and rtFhFtn-1(3) proteins against serum samples of infected patients with Fasciola.

Fig 4. Analysis of the reactivity folded recombinant proteins with their unfold forms.

Serum samples obtained from subjects with fascioliasis. Each recombinant proteins sample was assessed in triplicate.

Fig 5. ELISA assay showing the reactivity of rtFhSAP-2, rtFhFtn-1, rFhLAP, and combination.

(A) Scatter of rtFhSAP-2 ELISA absorbance value. (B) Scatter of rFhLAP ELISA absorbance value. (C) Scatter of rtFhFtn-1 ELISA absorbance value. (D) Scatter of combination ELISA absorbance value. (E) Scatter of E/S ELISA absorbance value, Horizontal line indicates the cutoff value established in both assays.