Metacercariae of C. sinensis were collected from freshwater fish, Pseudorasbora parva, which were obtained from an endemic area in China. They were then fed orally to rabbits and the adult flukes were recovered from the livers 12 weeks later. The recovered flukes were chopped and homogenized in ice, centrifuged at 15,000 rpm for 1 hour at 4℃ and the supernatant obtained (CA) was stored at -20℃ until required. The protein concentration of the CA was determined to be 3.69 mg/ml.

One hundred active moving worms which were recovered from rabbits were placed in 10 ml of phosphate buffered saline (PBS) at 37℃ for 15 hours. This PBS supernatant after centrifugation at 15,000 rpm at 4℃ was used as ESA at a protein concentration of 0.67 mg/ml.

A total of 509 samples of human clonorchiasis sera were obtained at an endemic locality in Heilongjiang, China. All infected subjects had a life-long history of consuming raw freshwater fish and were egg positive by stool examination. Healthy control serum samples were obtained from 163 healthy students at the Seoul National University. Sera were separated and stored at -70℃ Sera obtained from patients with other trematode infections were also used for comparison purposed; Opisthorchis viverrini (47 cases), Schistosoma japonicum (14 cases), Paragonimus westermani (20 cases), and Fasciola hepatica (3 cases). The sera of S. japonicum infection were obtained from Jiangxi, China and O. viverrini from Tailand, all subjects passed eggs in feces. The sera of P. westermani and F. hepatica infection were obtained from patients who were confirmed either parasitologically or pathologically.

ELISA was performed in polystyrene EIA microtitration 96-well flat-bottomed plates (Nunc Immunoplate I). Carbonate-bicarbonate buffer (pH 9.6) and phosphate buffered saline (pH 7.2) containing Tween 20 (PBST) were used for the coating procedures. For plate coating, 0.1 ml of antigen solution, protein concentration of which was 2 µg/ml, was added to each well. The plates were then sealed and incubated at 4℃ overnight. Blocking solution (1% bovine saline serum albumin in PBS, 0.5% NaN₃) was added after washing three times with PBST and incubating overnight. Subsequently, diluted (1:25) of test sera were added to the wells, and the plates were then incubated at 37℃ for one hour and washed with PBST. After shaking dry, 0.1 ml of diluted (1:50,000) horseradish peroxidase-labeled sheep IgG against human immunoglobulins (US Biochemical Cooperation) was added and incubated again for one hour at 37℃. The plates were then washed 3 times with PBST, and freshly prepared substrate solution (tetramethylbenzidine and 0.1% H₂O₂) was then added to each well. Following incubation at 37℃ for half an hour, the optical density was read at 450 nm using a Titertek Multiskan (Flow Laboratories). Samples with an absorbance of < 0.25 were deemed negative and those ≥ 0.25 as positive.

Fig. 1 and Table 1 show the distribution and the mean absorbances of the normal controls, and of the infected individuals. Mean ELISA absorbance for the CA of the serum samples of the controls was very low and individual absorbances showed little variation. Absorbances of the sera of clonorchiasis for CA much increased to mean level of 0.353 ± 0.058 (Table 1). Mean absorbances of the sera of other trematodiases for CA remained a little higher than 0.1.

The absorbances of serum samples of the controls for ESA were in quite similar level with those for CA. Mean absorbances of serum samples of clonorchiasis for ESA increased to 0.404 ± 0.129 and those of other trematodiases also increased a little (Table 1).

Sensitivity and specificity: Using the negativity/positivity criterion described above, the sensitivity of ESA-ELISA was 92.5%, which was higher than that of CA-ELISA, 88.2% (Table 2, P < 0.05, x²=5.52). Sera of the controls and those with F. hepatica infection showed no cross-reactions either as CA or ESA, but the sera of those infected with O. viverrini, S. japonicum, and P. westermani cross-reacted with both CA and ESA (Table 3). The results of our study show that ESA was more specific than CA as 93.1% versus 87.8%.