Immunolocalization Of Glutathione-S-transferase (SjGST) Of Schistosoma Japonicum In Eggs And Its Detection With Dot Immunogold Filtration Assay For Rapid Diagnosis Of Schistosomiasis

Objective: To investigate the expression and localization of glutathione- S-transferase (SjGST) in mature eggs of Schistosoma japonicum and explore the potential of rSjGST detection in dot immunogold filtration assay (DIGFA) for the diagnosis of schistosomiasis. Methods: The eggs were separated from liver tissues of the rabbits which had been infected with S.japonicum on day 42 after infection. The transcription and translation of SjGST gene in eggs were confirmed by RT-PCR and Western blotting with soluble egg antigen(SEA). Meanwhile, immunolocalization of SjGST within the mature eggs was performed with monoclonal antibody to rSjGST. Colloidal gold with an average diameter of 15 nm was generated with sodium citrate. Protein A absorbed to a red colloidal gold served as the antigen-antibody complex detecting reagent. The rSjGST antigens were subjected to the serological diagnosis in the patients with acute and chronic schistosomiasis japonica using DIGFA. The sensitivity and specificity of the recombinant antigens used in DIGFA (rSjGST-DIGFA) were compared in parallel with the SEA antigens used in DIGFA (SEA-DIGFA). In addition to the sera of patients with acute and chronic schistosomiasis, the detected samples include the sera of individuals with clonorchiasis and hookworm infections. Parallel tests were also performed using the rSjGST and SEA antigens in ELISA (rSjGST-ELISA and SEA-ELISA). Results: Consistent with the expected size, 670 bp of amplified product of RT-PCR was obtained and expression of SjGST in the developed eggs was recognized by Western blotting using monoclonal antibody to rSjGST. The SjGST was predominantly localized in lateral gland of miracidia in eggs. The samples including 102 cases of acute schistosomiasis, 207 cases of chronic schistosomiasis, 50 cases of Clonorchis sinensis, 73 cases of hookworms infection and 140 cases of normal control were tested using rSjGST-DIGFA, SEA-DIGFA, rSjGST-ELISA and SEA-ELISA respectively. The positive rates were 93.1 % in rSjGST-DIGFA and 98.0 % in SEA-DIGFA in detection of acute schistosomiasis. Statistical analysis revealed no significant difference in sensitivity (p>0.05) between both recombinant and crude antigens; the positive rates in detection of chronic schistosomiasis were 80.7 % in rSjGST-DIGFA and 84.1 % in SEA-DIGFA, no significant difference of sensitivity (p>0.05) in those cases was noted. The fales positive reaction was found to be 3.6 % in rSjGST-DIGFA and 1.4 % in SEA-DIGFA when detected in 140 cases of normal control sera but no statistically significant difference was found (p>0.05). 90.2 % and 99.0 % were found to be positive (p>0.05) when rSjGST-ELISA and SEA-ELISA were subjected to the detection in 102 cases of acute S. japonicum infection; and 82.1 % and 87.4 % in 207 cases of chronic infection (p>0.05); 2.1 % and 2.1 % of fales positivity were seen in 140 cases of normal control (p>0.05). 14.0 % and 8.0 % of cross-reactions were observed between rSjGST-DIGFA and SEA-DIGFA for detecting the sera of patients with Clonorchis sinensis and 6.8 % and 4.1 % in the cases of hookworm infection. There was no significant difference of cross-reaction in two parasitic infections (p>0.05, p>0.05) with the two tests. 10.0 % and 10.0 % of cross-reactions were observed between rSjGST-ELISA and SEA-ELISA for detecting the sera of patients with Clonorchis sinensis and 6.8 % and 4.1 % in the cases of hookworm infection. There was also no significant difference of cross-reaction in two parasitic infections (p>0.05, p>0.05) with the two tests Conclusion: Our results demonstrate that using recombinant SjGST as antigens in DIGFA (rSjGST-DIGFA) for the detection of sera of both acute and chronic schistosomiasis has similar sensitivity and specificity to the soluble eggs antigens (SEA) in DIGFA (SEA-DIGFA). The results indicated that the rSjGST may be a promising tool for field serological surveys in the epidemic areas of schistosomiasis. The rSjGST antigen, together with DIGFA, is easy to standardize, economic to produce, simple to carry out, and has the potential of practical use for the immunodiagnosis of schistosomiasis.