Plerocercoid larvae were collected from naturally infected snakes, Rhabdophis tigrinus tigrinus. The collected spargana were chopped up and homogenized in ice-cold PBS and centrifuged at 13,000 rpm for 30 min at 4℃. The supernatant was filtered using 0.20 µm Acrodisc® Syringe Filters (Pall Corporation, Ann Arbor, Missouri, USA). The concentration of crude antigen was measured using the BCA™ Protein Assay Kit (Pierce, Rockford, Illinois, USA), and the crude antigen was stored at -70℃ until use.

A total of 132 human serum samples of parasitologically and clinically proven cases were used for the evaluation of ELISA. Positive control serum samples (n=20) were obtained from patients with sparganosis that had been confirmed by either clinically or surgically. Negative control sera (n=65) were collected from healthy Korean subjects without any parasitological infections. For evaluation of cross-reactions, 47 serum specimens from patients with other parasitic infections were also used: these infections included toxocariasis (n=10), clonorchiasis (n=19), cysticercosis (n=5), paragonimiasis (n=8), and anisakiasis (n=5).

Serum specimens from experimentally infected (n=10) and uninfected mice (n=10) were also used for evaluation of ELISA. Serum samples were collected from mice after 2 months of oral infection with sparganum scolices.

The gene encoding the mature domain of a sparganum cysteine protease was amplified by PCR using the sparganum cDNA. The primers were designed based on previously reported full-length cDNA sequences of Spirometra erinacei plerocercoid cysteine protease (GenBank accession no. D63670.1) [17] as follows: forward primer, 5'-GAGAGGTACCCTTCCCGACAGCGTAAACTGGCGC-3' and reverse primer, 5'-GAGAAAGCTTCACGGTTGGATAGCTTGCCATGCT-3'. Restriction sites for Kpn I and Hind III were added at the 5' ends of the forward and reverse primers, respectively. The PCR reaction was performed in a 50 µl reaction volume that contained 5 µl of 10x PCR buffer (Takara Shuzo Col, Kyoto, Japan), 0.5 µM each primer, 200 µM dNTP, 1.5 U of Taq polymerase, and 2 µl of 1:5 diluted cDNA. The PCR amplification reaction consisted of a Taq DNA polymerase activation step at 95℃ for 5 min followed by 35 cycles of denaturation at 95℃ for 30 sec, annealing at 68℃ for 30 sec, and extension at 72℃ for 60 sec. The amplified PCR products were purified and digested with restriction enzymes. The resulting insert was ligated into the pQE30 expression vector, which was then transformed into E. coli M15 (pREP4) competent cells (Qiagen, Hilden, Germany) by standard methods. Transformants containing the pQE30 plasmid with the insert were screened by restriction analysis and confirmed by sequencing.

Expression of the recombinant S. erinacei plerocercoid cysteine protease (rSepCp-1) was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM at 37℃ for 4 hr. Cells were harvested by centrifugation at 4,000 g for 20 min. The rSepCp-1 protein expressed as inclusion bodies was solubilized completely with 8 M urea in 10 mM Tris-Cl and 100 mM NaH₂PO₄ buffer (pH 8.0). Then, the histidine-tagged protein was purified using a nickel-nitrilotriacetic acid (Ni-NTA) agarose column according to the manufacturer's instructions (Qiagen).

The recombinant protein was electrophoresed on 12% acrylamide gels. After electrophoresis, proteins were stained with Coomassie blue or transferred to nitrocellulose membranes (Millipore, Bedford, Massachusetts, USA). For immunoblotting analysis, membranes were blocked with 0.05% Tween-20 in PBS (PBST) containing 5% skim milk and were reacted with a mouse anti-histidine monoclonal antibody (1:4,000) (R&D Systems Inc., Minneapolis, USA), sparganum-positive human or mouse serum samples or negative serum samples (1:4,000) for 2 hr at room temperature. After 3 washes with PBST, membranes were incubated with peroxidase-conjugated secondary antibody against mouse IgG (1:4,000) (DakoCytomation, Glostrup, Denmark) or human IgG (1:4,000) (DakoCytomation) for 1 hr at room temperature, followed by detection with an immunoblotting detection reagent (West-Zol® Plus, Intron Biotechnology, Sungnam, Korea).

Polystyrene microtiter plates (Costar, Corning, New York, USA) were coated with 0.5 µg/well (100 µl/well) of rSepCp-1 protein or sparganum crude extract (SeC) diluted in 0.1 M carbonate/bicarbonate buffer, pH 9.6, and incubated at 4℃ overnight. After washing 3 times with PBST, plates were blocked with 1% bovine serum albumin for 1 hr at 37℃. After washing, 100 µl of human or mouse serum samples diluted 1:500 were loaded into each well of the plates, and the plates were incubated for 2 hr at 37℃. Then, 100 µl of horseradish peroxidase-conjugated goat anti-human IgG (1:10,000) (ICN Pharmaceuticals Inc., Aurora, Ohio, USA) or rabbit anti-mouse IgG (1:5,000) (DakoCytomation) was loaded into each well. After incubation for 1 hr at 37℃, the reaction was developed with 1-Step™ Ultra TMB-ELISA substrate (Pierce) at room temperature, and the absorbances were read at 450 nm. All samples were run in duplicate. The cut-off values were defined as the mean absorbance plus 3 SDs of the values obtained from the sera of healthy negative controls.

The statistical significance of ELISA results was evaluated by the Student's t-test, and differences were considered significant when the P-values were less than 0.05.