应用抗SjP38的单克隆抗体建立血吸虫感染的胶体金免疫层析检测体系

A colloidal gold immunochromatographic assay for detecting p38 antigen of Schistosome japonicum

目的建立一种检测日本血吸虫感染的胶体金免疫层析方法。方法将已获得的单抗用protein-G亲和层析纯化,通过间接ELISA测定每株单抗的亲和常数。按改良过碘酸钠标记法,8株单抗标记辣根过氧化物酶(HRP)后进行不同株单抗配对,选出亲和力高、稳定性强的单抗配对组合。胶体金标记4株抗体,选择最佳反应模式,建立胶体金免疫层析方法,并对弓形虫感染鼠血样、血吸虫感染前和感染后2、3、4、5、6周的鼠血样进行检测。结果8株抗SjP38单克隆抗体经纯化后纯度达95%以上,其亲和常数介于1×10-8mol/L^2.82×10-10mol/L。根据配对实验,1A6与9H6、1A6与9G7、6G12与9H6、6G12与9G7以及9H6与9G7为最佳配对组合。标记1A6、6G12、9H6与9G7这4株单抗后筛选出最佳反应模式,即以9G7为包被抗体,包被浓度为2.5μg/μl;当1A6为金标抗体,抗体稀释浓度为1:4时,检测效果最佳。对rSjP38的最低检测量为12.5ng/ml。抗检测感染3、4、5、6周小鼠血清,阳性率分别为40%、50%、60%、80%,而弓形虫感染鼠和血吸虫感染前小鼠血样的检测结果为100%阴性。结论通过抗体配对、最佳包被量和金标抗体稀释倍数条件的优化,建立了快速、简便、特异的胶体金免疫层析方法,自制的rSjP38试纸条能检测血吸虫早期感染鼠血清中的循环抗原SjP38。

Objective To establish a colloidal gold immunochromatographic assay (GICA) for detecting Schistosome japonicum (Sj). Methods Eight monoclonal antibodies (mAbs) against Sj p38 antigen (1A6, 3C4, 3D12, 6F10, 6G12, 9H6, 9G7 and A5H) prepared previously were purified by protein-G affinity chromatography. The affinity constant (Kaff) was determined by indirect enzyme-linked immunosorbent assay. All the mAbs were labeled with horseradish peroxidase by sodium oxidation method and the mAb pairs with high affinity and stability were identified according to their optical density at 450 nm (OD450). Four mAbs (1A6, 6G12, 9H6 and 9G7) were chosen for colloidal gold labeling. GICA was then performed by further optimization of the labeling and the conditions were determined. The sera of mice at different infection stages were examined with GICA dipstick, with the sera collected before infection and those of Toxoplasma gondii-infected mice as negative controls. Results The purity of the 8 mAbs was higher than 95% with Kaff ranging from 2.8×10-10 to 1×10-8 mol/L. 9G7 coating (2.5 mg/ml) as the capture antibody and detection with 1A6 (diluted at 1:4) as the labeling antibody was determined as the best reaction model. With this combination, the positivity rates of the detection were 40%, 50%, 60% and 80% for mouse sera collected at 3, 4, 5 and 6 weeks after Schistosome japonicum infection, respectively, without positive results for the negative control samples. Conclusion GICA established in this study is characterized by simplicity, rapidity and good sensitivity, and the prepared rSjP38 dipstick can test the circulating antigen SjP38 in early stage of infection.