Expression, purification and in vitro refolding of the recombinant truncated Saposin-like protein 2 antigen for development of diagnosis of human fascioliasis

Mirzadeh, Abolfazl; Valadkhani, Zarrintaj; Yoosefy, Asiyeh; Babaie, Jalal; Golkar, Majid; Rastaghi, Ahmad Reza Esmaeili; Kazemirad, Elham; Ashrafi, Keyhan

doi:10.1016/j.actatropica.2017.03.002

Cited by 10 publications

(15 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Furthermore, it has been shown that obtaining large quantities of highly pure recombinant F . hepatica antigens with correct folding play a vital role in improvement of antigenicity and accuracy in serodiagnosis methods [ 14 , 27 ]. In this line, we evaluated the immunodiagnostic potential of tFhSAP-2, tFh-Ftn-1 and FhLAP recombinant antigens, alone or in combination, for the detection of IgG antibodies in fascioliasis by ELISA.…”

Section: Discussionmentioning

confidence: 99%

“…Accordingly, we expressed recombiant proteins under different conditions along with the diverse expression vectors and strains to obtain optimized recombinant proteins, ( Fig 1 ). After proteins isolation with the highest purity ( Fig 2 ), refolding process was performed using dialysis process in the presence of chemical additives, and reduced/oxidized glutathione ( in vitro ), which is known as a highly efficient method [ 14 , 36 ]. The results proved that refolding proteins are highly more efficient in diagnosis of IgG antibody compared to unfolded proteins ( Table 2 and Fig 4 ).…”

Section: Discussionmentioning

confidence: 99%

“…The pellet related to each protein were achieved from 250 ml culture media and suspended in 14 ml isolation buffer (350mM NaCl, 5 mM EDTA 50mM Tris HCl, 1 mM PMSF), the pH of buffer for rtFhSAP-2 and FhLAP proteins was 8 and for rtFhFtn-1was 7. The process of isolation as well as washing of IBs were performed the same as Mirzadeh et al [ 14 ]. The obtained pellet from washing of IBs, were suspended in 25 ml of binding buffer (Tris–HCl 30 mM, NaCl 500 mM, Urea 8 M and imidazole 2 mM, pH 8.0) (pH of binding buffer for purification of FhFtn-1 was 7), containing dithiothreitol (DTT) 10 mM and phenyl methyl sulfonyl fluoride (PMSF) 5mM.…”

Section: Bacterial Strains Plasmidsmentioning

confidence: 99%

“…More recently, several recombinant proteins have been evaluated in an ELISA test to detect IgG antibodies [ 14 , 15 ]. In this line, F .…”

Section: Introductionmentioning

confidence: 99%

“…In this line, F . hepatica saposin-like protein 2 (FhSAP-2) has been suggested as one of the ideal antigens for development of ELISA kits that exhibited a highly strong antibody response in the acute and chronic phase of infection [ 14 , 15 , 17 ]. Moreover, the F .…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

et al. 2018

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Bacterial Strains Plasmidsmentioning

confidence: 99%

“…More recently, several recombinant proteins have been evaluated in an ELISA test to detect IgG antibodies [ 14 , 15 ]. In this line, F .…”

Section: Introductionmentioning

confidence: 99%