比较华支睾吸虫不同时段排泄/分泌产物（CsESP）的免疫原性，评估其用于诊断华支睾吸虫病的价值。体外培养华支睾吸虫成虫，分别收集培养0~24 h和24~48 h的培养液并混合，制备成CsESP-1和CsESP-2抗原。小鼠随机分为CsESP-1组、CsESP-2组、佐剂组和PBS组4组，使用对应的抗原进行皮下免疫，并于免疫第0、2、4、6周采集血液制备免疫血清，ELISA法检测免疫小鼠血清中CsESP抗体滴度和IgG、IgG1水平。以CsESP-1和CsESP-2抗原ELISA检测健康者（Cs^-）和华支睾吸虫感染者（Cs⁺）血样中CsESP特异性IgG及亚类水平，使用SPSS 27.0软件生成敏感度、特异度和约登指数，绘制受试者工作特征（ROC）曲线并计算ROC曲线下面积（AUC），根据AUC评价抗原的诊断效果。取Cs^-、Cs⁺、粪类圆线虫感染者、巨片形吸虫感染者和疟疾患者血样，ELISA检测交叉反应。两组比较采用t检验，AUC的比较采用配对ROC分析。ELISA结果显示，免疫第6周CsESP-1组和CsESP-2组小鼠血清的CsESP抗体滴度分别为1:102 400和1:400，CsESP-1的抗体滴度优于CsESP-2。CsESP-1组小鼠在免疫第6周的特异性IgG和IgG1 A₄₅₀值分别为0.926和1.803，均高于佐剂组的0.147和0.080（t = 5.805、5.643，均P < 0.05）。CsESP-2组小鼠在免疫第2、4、6周的特异性IgG A₄₅₀值分别为0.279、0.437、0.523，均高于佐剂组的0.133、0.127、0.142（t = 2.906、5.286、6.83，P < 0.05、0.05、0.01）；在第4、6周的IgG1 A₄₅₀值为0.225和0.332，均高于佐剂组的0.095和0.083（t = 3.423、2.485，均P < 0.05）。Cs⁺血样中CsESP-1特异性IgG、IgG2和IgG4的A₄₅₀值分别为0.817、0.274和0.524，均高于Cs^-血样的0.456、0.102和0.103（t = 7.184、2.720、5.634，均P < 0.01）；CsESP-2特异性IgG、IgG1、IgG2和IgG4的A₄₅₀值分别为0.783、0.132、0.233和0.465，均高于Cs^-血样的0.460、0.068、0.074和0.082（t = 4.768、2.519、2.634、4.991，P < 0.01、0.05、0.05、0.01）。CsESP-1抗原检测Cs⁺血样IgG4的综合效果最优，敏感度、特异度和准确度分别为89.2%、86.5%和87.9%；CsESP-2抗原检测Cs⁺血样IgG4的综合效果也最优，敏感度、特异度和准确度也分别为89.2%、86.5%和87.9%。ROC曲线显示，CsESP-1抗原检测IgG1和IgG2均具有一定的诊断价值（均AUC > 0.7），检测IgG和IgG4均具有较高的诊断价值（均AUC > 0.9）；CsESP-2抗原检测IgG、IgG1和IgG2均具有一定的诊断价值（均AUC > 0.7），检测IgG4具有较高的诊断价值（AUC > 0.9）。配对ROC分析显示，CsESP-1抗原对IgG和IgG1的诊断效能高于CsESP-2抗原（z = 3.515、2.149，P < 0.01、0.05）。CsESP-1和CsESP-2抗原与粪类圆线虫感染者、巨片形吸虫感染者和疟疾患者血样的IgG均无交叉反应。本研究制备的CsESP-1和CsESP-2抗原均具有良好的免疫原性，可作为诊断华支睾吸虫病的候选抗原。

To compare the immunogenicity of excretory/secretory products of Clonorchis sinensis (CsESP) at different intervals and evaluate their diagnostic value for clonorchiasis. Adult C. sinensis were cultured in vitro, and the culture supernatants collected 0-24 hours and 24-48 hours were mixed to prepare CsESP-1 and CsESP-2 antigens, respectively. Mice were randomly divided into 4 groups: CsESP-1, CsESP-2, adjuvant and PBS groups. Mice in each group were subcutaneously immunized with the corresponding antigen. Blood samples were collected at weeks 0, 2, 4 and 6 post-immunization to prepare immune sera. ELISA was used to measure the antibody titers against CsESP and levels of IgG and IgG1. Additionally, ELISA was employed to detect the levels of CsESP-specific IgG and its subclasses in blood samples from healthy individuals and those infected with C. sinensis. SPSS 27.0 software was used to calculate sensitivity, specificity and Youden’s index. The receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) were computed to evaluate the diagnostic efficacy of the antigens. Blood samples from healthy individuals (Cs^-) and those infected with C. sinensis (Cs⁺), Strongyloides stercoralis, Fasciola gigantica and malaria patients were tested for cross-reactivity using ELISA. Comparisons between two groups were conducted using the t-test, while paired ROC analysis was used for AUC comparisons. ELISA results showed that at week 6 post-immunization, the antibody titers against CsESP in the sera of mice in the CsESP-1 and CsESP-2 groups were 1:102 400 and 1:400, respectively, indicating superior immunogenicity of CsESP-1. At week 6 post-immunization, the A₄₅₀ values for specific IgG and IgG1 in the sera of mice in the CsESP-1 group were 0.926 and 1.803, respectively, both significantly higher than those in the adjuvant group (0.147 and 0.080, respectively) (t = 5.805, 5.643; both P < 0.05). In the CsESP-2 group, the A₄₅₀ values for specific IgG at weeks 2, 4 and 6 post-immunization were 0.279, 0.437 and 0.523, respectively, all higher than those in the adjuvant group (0.133, 0.127, 0.142) (t = 2.906, 5.286, 6.83; P < 0.05, 0.05, 0.01). The A₄₅₀ values for IgG1 in the CsESP-2 group at weeks 4 and 6 post-immunization were 0.225 and 0.332, respectively, both higher than those in the adjuvant group (0.095, 0.083) (t = 3.423, 2.485; both P < 0.05). In Cs⁺ blood samples, the A₄₅₀ values for CsESP-1-specific IgG, IgG2 and IgG4 were 0.817, 0.274 and 0.524, respectively, all higher than those in Cs^- blood samples (0.456, 0.102 and 0.103, respectively) (t = 7.184, 2.720, 5.634; all P < 0.01). For CsESP-2, the A₄₅₀ values in Cs⁺ blood samples for specific IgG, IgG1, IgG2 and IgG4 were 0.783, 0.132, 0.233 and 0.465, respectively, all higher than those in Cs^- blood samples (0.460, 0.068, 0.074 and 0.082, respectively) (t = 4.768, 2.519, 2.634, 4.991; P < 0.01, 0.05, 0.05, 0.01). The CsESP-1 antigen demonstrated the best overall performance in detecting IgG4 in Cs⁺ blood samples, with sensitivity, specificity and accuracy of 89.2%, 86.5% and 87.9%, respectively. Similarly, the CsESP-2 antigen showed the same sensitivity specificity and accuracy (89.2%, 86.5% and 87.9%, respectively) for detecting IgG4 in Cs⁺ samples. ROC curve analysis revealed that the CsESP-1 antigen had moderate diagnostic value for detecting IgG1 and IgG2 (both AUC > 0.7) and high diagnostic value for detecting IgG and IgG4 (both AUC > 0.9). The CsESP-2 antigen exhibited moderate diagnostic value for detecting IgG, IgG1 and IgG2 (all AUC > 0.7) and high diagnostic value for detecting IgG4 (AUC > 0.9). Paired ROC analysis indicated that the diagnostic efficacy of the CsESP-1 antigen for IgG and IgG1 was superior to that of the CsESP-2 antigen (z = 3.515, 2.149; P < 0.01, 0.05). Neither the CsESP-1 nor CsESP-2 antigens showed cross-reactivity with IgG in blood samples from individuals infected with S. stercoralis, F. gigantica or malaria patients. In conclusion, both the CsESP-1 and CsESP-2 antigens prepared in this study exhibited good immunogenicity and could serve as candidate antigens for the diagnosis of clonorchiasis.