| Objective: After 42-day infection with cercarie of Schistosoma japonicum, the eggs were obtained from the liver tissues of infected New-Zewland rabbits, and then soluble egg antigen(SEA) were prepared. SEA was used to immunize the rabbit and Roman hens to generate immunoglobulin G (IgG) and Y (IgY) against SEA, respectively. The specificity of IgY was tested by Western blotting and biochemical properties were characterized by indirect ELISA. The double antibody sandwich ELISA assay, based on IgG and IgY antibodies, were established, which was subsequently used for SEA detection. And the sensitivity and specificity of this assay were also analyzed. Methods: 4, 500 cercarie of S. japonicum-positive snails which were purchased from Jiangsu Institute of Parasitic Diseases Contral, were used to infect the New-Zewland rabbits. Forty-two days postinfection, the eggs were isolated from the liver tissues in order to prepare SEA. Each of the egg-laying Roman hens (6 months old) was immunized with 450μg SEA and the eggs were collected to prepare anti-SEA IgY antibody. The specificity of IgY was analyzed by Western blotting, and the titre of IgY and its biochemical properties were evaluated by indirect ELISA. Meanwhile, the rabbits were immunized with SEA to get anti-SEA IgG. Double antibody sandwich ELISA was established, with anti-SEA IgG for antigen capture and IgY for detection. The detection system, such as concentration of capturing IgG and detecting IgY, were optimized and used in the subsequent experiments. With optimized ELISA method, sera from 443 cases, including 68 patients with acute and 189 with chronic schistosomiasis, and 100 normal human controls, were tested, and the sensitivity, specificity and cross-reaction were also evaluated, respectively. Results: The concentration of obtained SEA was 300mg/L. SDS-PAGE analysis of the anti-SEA IgY antibodies showed that the molecular weight of IgY was the same as expected, and the results of indirect ELISA and Western blotting indicated that IgY was specific against SEA. We also found that the concentration of IgY was 5.04mg/ml and the tire was 1:1×105. IgY was found to be heat- and low pH-resistant, and could not be degraded by pepsin digestion at 37℃. The double antibody (IgG-IgY) sandwich ELISA system was established for diagnosis of acute and chronic schistosomiasis. We found that the positive detection rates of this method for acute and chronic schistosomiasis achieved 79.4% and 64.4%, and the specificity was 92%. The rates of cross-reaction with paragonimiasis, clonorchiasis, and hockworm infections were 20%, 20.8%, and 14.8%, respectively. With the use of IgY-based ELISA method, we found that 15%,45%,90% and 100% of the slightly infected mice (5 cercarie per mouse) turned to be negative by detection of circulating SEA when treated by Praziquantel at 2, 4, 6 and 12 months postinfection, respectively; For the moderately infected mice (15 cercarie per mouse), SEA was undetectable in 5%,25%,40% and 70% of mice respectively after treatment at 2, 4, 6 and 12-months postinfection; With the same treatment, 0%,10%,30% and 60% of the heavily infected mice (35 cercarie per mouse) turned negative of SEA detection. Conclusion: We have obtained the anti-SEA IgY antibody, established and optimized the double antibody sandwich ELISA system to detect SEA of S. japonicum from the sera of acute and chronic patients with schistosomiasis and normal persons. The cross-reaction with sera of paragonimiasis, clonorchiasis and hookworm infections were also investigated. Our results demonstrated that the IgY-ELISA method was effective in evaluating the therapeutic effect in different parasitic burden of infected mice, which provides a novel technique for clinical diagnosis of S. japonicum infection. |
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