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目的:建立双抗体夹心ELISA法检测日本血吸虫硫氧还蛋白(Thioredoxin,Trx)。方法:用重组日本血吸虫Trx(rTrx)蛋白免疫BALB/c小鼠,筛选高滴度、高特异性的单克隆抗体建立双抗体夹心ELISA法。通过检测日本血吸虫排泄-分泌物(excretorysecretions,ES)与rTrx的浓度评价该方法的敏感性;通过对健康人血清的检测确定其特异性;通过对布氏姜片吸虫病、华支睾吸虫病、卫氏并殖吸虫病、囊虫病患者血清进行交叉反应试验,评价该方法的特异性。结果:获得2株稳定分泌抗rTrx蛋白单克隆抗体的杂交瘤细胞株,命名为McTrx1和McTrx2。以McTrx1为包被抗体,HRP-McTrx2为酶标抗体,建立的双抗体夹心ELISA可检测出ES的最低浓度为4.8μg/ml,检测出rTrx的最低浓度为1.2μg/ml。该方法的特异性为96%。结论:以抗rTrx蛋白单克隆抗体McTrx1与McTrx2为基础建立的双抗体夹心ELISA法具有较高的特异性。
Objective: To establish a double antibody sandwich ELISA for the detection of Thioredoxin (Trx) in Schistosoma japonicum. Methods: BALB / c mice were immunized with trx (rTrx) protein of Schistosoma japonicum and screened for high titer and high specificity of monoclonal antibodies to establish a sandwich ELISA. The sensitivity of this method was evaluated by detecting the excretory secretion (ES) and rTrx concentrations of Schistosoma japonicum; its specificity was determined by detection of healthy human serum; , Paragonimus westermani disease, cysticercosis patients serum cross-reactivity test to evaluate the specificity of the method. Results: Two hybridoma cell lines stably secreting anti-rTrx monoclonal antibody were obtained and named as McTrx1 and McTrx2. Using McTrx1 as coated antibody and HRP-McTrx2 as enzyme-labeled antibody, the established double-antibody sandwich ELISA can detect the lowest ES concentration of 4.8μg / ml and the lowest rTrx concentration of 1.2μg / ml. The specificity of this method is 96%. Conclusion: Double antibody sandwich ELISA based on the anti-rTrx monoclonal antibodies McTrx1 and McTrx2 has high specificity.