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目的应用华支睾吸虫PPMPⅠ型抗原重组Cs2蛋白(rCs2)建立2种华支睾吸虫病免疫学检测方法,并进行初步评价。方法以可溶性rCs2作为诊断抗原,建立间接ELISA检测方法,并研制胶体金免疫层析检测(GICA)动力流体型试条,检测血清中的特异性抗体。采用ELISA法和GICA试条共检测35例华支睾吸虫虫卵粪检阳性患者、15例日本血吸虫病患者、15例卫氏并殖吸虫病患者和13例猪囊尾蚴病患者的血清,以及33份健康人血清。为进一步评价诊断价值,8只新西兰兔各感染600个华支睾吸虫囊蚴,随机均分为感染组和治疗组,感染第56天,治疗组各兔给予吡喹酮治疗[150 mg/(kg.d)×2 d],用ELISA法和GICA法检测两组兔0~44周抗rCs2特异性抗体的消长。结果 ELISA法的敏感性为71.4%(25/35),特异性为93.4%(71/76),总符合率为86.5%(96/111),与日本血吸虫病、卫氏并殖吸虫病和猪囊尾蚴病患者血清的交叉反应阳性率分别为1/15、1/15和1/13。GICA试条检测法的敏感性为85.7%(30/35),特异性为92.1%(70/76),总符合率为90.1%(100/111)。经ELISA检测,感染组兔自感染后第4周抗rCs2特异性抗体水平开始上升,于第6周达到高峰,随后快速下降,至第20周已呈较低水平;治疗组的特异性抗体消长趋势同感染组,同一时点的2组实验兔的抗体水平差异无统计学意义(P>0.05)。GICA试条检测结果表明,2组兔血清均仅在4~16周检出阳性。结论以PPMPⅠ型抗原rCs2建立的间接ELISA方法和GICA动力流体型试条对华支睾吸虫病具有较好的诊断价值。
Objective To establish two immunocompetent detection methods of Clonorchis sinensis by using CsA1 gene of Clonorchis sinensis PPMP Ⅰ gene and make a preliminary evaluation. Methods Soluble rCs2 was used as a diagnostic antigen to establish an indirect ELISA method. Colloidal gold immunochromatographic test (GICA) dynamic fluid type test strips were developed to detect serum specific antibodies. ELISA and GICA test strips were used to detect serums of 35 cases of Clonorchis sinensis egg ovulation positive patients, 15 cases of Japanese schistosomiasis, 15 cases of Paragonimiasis wiht and 13 cases of cysticercosis patients, and 33 healthy human serum. To further evaluate the diagnostic value, eight New Zealand rabbits each infect 600 Clonorchis sinensis metacercariae and were randomly divided into infection group and treatment group. On the 56th day of infection, the rabbits in the treatment group were treated with praziquantel [150 mg / ( kg.d) × 2d]. The growth of anti-rCs2-specific antibodies at 0 ~ 44 weeks in both groups were detected by ELISA and GICA. Results The sensitivity of ELISA was 71.4% (25/35), specificity was 93.4% (71/76), and the total coincidence rate was 86.5% (96/111). The sensitivity and specificity of ELISA were significantly higher than those of Japanese schistosomiasis, Paragonimus westermani, The positive rate of cross-reaction of serum of patients with cysticercosis was 1 / 15,1 / 15 and 1/13 respectively. The sensitivity of GICA test strip was 85.7% (30/35), the specificity was 92.1% (70/76), and the total compliance rate was 90.1% (100/111). The level of anti-rCs2-specific antibody in infected rabbits increased from the 4th week after infection to the peak at 6th week after infection, and then rapidly decreased to the lowest level at the 20th week. The specific antibodies in the treatment group showed a decrease The trend of the same infection group, the same time the two experimental rabbits antibody levels showed no significant difference (P> 0.05). GICA test strip test results showed that two groups of rabbit serum were positive only in 4 to 16 weeks. Conclusion The indirect ELISA method and GICA dynamic fluid type test set up by PPMP Ⅰ type antigen rCs2 have good diagnostic value to Clonorchis sinensis.