胱抑素C的IgG-和IgY-ELISA检测体系的建立与评价  

Establishment and evaluation of IgG-and IgY-ELISA detection system for Cystatin C

作　　者：蒋洪娇[1] 李敏惠 邹强 王琦[1] 吴永琴[1] 吴啟洪 邓未[1] 刘雯雯[1] 杨艳玲[1] 代爽[1] 杨平[3] 

机构地区：[1]成都医学院生物医学系,成都610083 [2]成都医学院科研实验中心,成都610083 [3]成都医学院生物化学教研室,成都610083

出　　处：《成都医学院学报》2012年第3期386-389,共4页Journal of Chengdu Medical College

基　　金：国家自然科学基金(NO:81273530);四川省教育厅青年基金(NO:10ZB095);成都医学院综合性;设计性实验项目(NO:ZH201114);成都医学院横向课题(NO:10H018);成都医学院科研基金(NO:CYZ11-005)

摘　　要：目的建立适用于检测人胱抑素C(Cystatin C)的IgG与IgY双抗体夹心ELISA检测体系。方法以人Cystatin C作为抗原免疫产蛋的罗曼鸡和新西兰兔,纯化抗Cystatin C的鸡抗体IgY和兔抗体IgG,并分别建立三种ELISA夹心检测体系,I:捕捉抗体IgG+检测抗体IgY+抗鸡IgY酶标抗体;Ⅱ:捕捉抗体IgG+检测抗体IgY+兔F(ab')2抗鸡酶标抗体;Ⅲ:捕捉抗体IgY+检测抗体IgG+抗兔IgG酶标抗体。通过比较Cystatin C的最低检测浓度和阴性对照的OD值评价三种夹心法的灵敏度和特异性。结果体系I的Cystatin C最低检测浓度为7.5ng/mL(阴性对照OD值为0.737),体系Ⅱ的Cystatin C最低检测浓度为13ng/mL(阴性对照OD值为0.313),体系Ⅲ的Cystatin C最低检测浓度为10ng/mL(阴性对照OD值为0.31)。结论成功建立了Cystatin C的双抗体夹心ELISA检测体系,体系I的灵敏度最高但其特异性差,体系Ⅱ和Ⅲ的检测灵敏度和特异性均较好,可以用于Cystatin C的检测。Objective To establish double-antibodies sandwich ELISA detection system based on IgG and IgY antibodies against Cystatin C. Methods Hens and new-zealand rabbits were immunized with Cystatin C as antigen and the Cystatin C polyclonal antibodies IgY and IgG were purified separately. Three kinds of ELISA detection systems;Ⅰ： capture antibody IgG ＋ detection antibody IgY ＋ enzyme labelled antibody against IgY;Ⅱ ： capture antibody IgG＋ detection antibody IgY＋ enzyme labelled antibody F （ab） 2 against IgY ; Ⅲ ： capture antibody IgY ＋ detection antibody IgG＋ enzyme labelled antibody against IgG, were established. The sensitivity and specificity of these three kinds of sandwich assays were evaluated by the comparison of the Cystatin C minimum detection concentrations and the OD values of the negative controls. Results The Cystatin C minimum detection concentration of system I was 7. 5 ng/mL（OD value of the negative control was 0. 737）. The Cystatin C minimum detection concentration of system Ⅱ was 13 ng/mL（OD value of the negative control was 0. 313）. The Cystatin C minimum detection concentration of system Ⅲ was 10 ng/mL（OD value of the negative control was 0. 31）. Conclusions Double-antibodies sandwich ELISA detection system based on IgG and IgY antibodies against Cystatin C was created successfully. System Ⅱ and system Ill can be used for the detection of Cystatin C because of the better detection sensitivity and specificity than system I.

关 键 词：IGY抗体 IGG抗体 胱抑素C 酶联免疫吸附试验 

分 类 号：R743.305[医药卫生—神经病学与精神病学]