α-唾液淀粉酶胶体金免疫渗滤法的建立及其在中药酶学作用研究中的应用  

Development of a dot immunogold filtration assay for salivary alpha-amylase and its application in enzymological research on traditional Chinese medicine

作　　者：金旻逸 丁越 陈静雯 兰金帅 李俊松 钟高仁[3] 张彤 JIN Min-yi;DING Yue;CHEN Jing-wen;LAN Jin-shuai;LI Jun-song;ZHONG Gao-ren;ZHANG Tong(Experiment Center for Teaching and Learning,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;不详)

机构地区：[1]上海中医药大学教学实验中心,上海201203 [2]复旦大学附属妇产科医院,上海200090 [3]复旦大学药学院,上海200032

出　　处：《中国生物制品学杂志》2021年第3期340-348,共9页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2019ZX09201004-002);上海市中医药事业发展三年行动计划[ZY(2018-2020)-CCCX-2001-04];上海市学术带头人(18XD1403700);上海中医药大学预算内科研项目(2019LK072)。

摘　　要：目的建立α-唾液淀粉酶(salivary alpha-amylase,sAA)胶体金免疫渗滤法,制备胶体金免疫渗滤试纸,并将该试纸用于中药白术及葛根的酶学作用研究。方法采用胶体金标记技术,筛选最佳标记pH值和抗体标记量,制备金标抗体复合物,再利用纸基免疫渗滤技术,设计单因素试验,优化包被液种类、包被条件、封闭液种类、封闭条件、洗涤液种类、抗原抗体最佳工作浓度、包被抗原与金标抗体反应条件、待测样品与金标抗体反应条件共8个影响因素,建立胶体金免疫渗滤检测法,同时对免疫试纸的灵敏度和初步稳定性进行验证,并利用该试纸检测不同浓度中药白术和葛根提取液对sAA与其抗体特异性结合活性的促进或抑制作用。结果胶体金标记抗体的最佳pH值为8.5,最佳抗体稀释比例为1∶125,在此条件下制备的金标抗体复合物稳定性良好,活性效价为1∶384。sAA胶体金免疫渗滤法的最适反应条件为:以含8%甲醇的PBS缓冲液(0.01 mol/L,pH 7.4)为包被液稀释sAA至10μg/mL,37℃包被15 min;以含2%BSA的PBS缓冲液(0.01 mol/L,pH 7.4)为封闭液,37℃封闭15 min;以含0.4%Tween-20的PBS缓冲液(0.01 mol/L,pH 7.4)为洗涤液对NC膜进行洗涤;待测样品与稀释比例为1∶1的金标抗体于37℃下提前反应15 min,加入NC膜后25℃条件下再反应20 min,洗涤后根据斑点显色情况判断实验结果。制备的胶体金免疫渗滤试纸灵敏度为5μg/mL,稳定性良好。中药酶学作用研究结果显示,白术可促进sAA与其抗体的特异性结合,而葛根具有抑制作用,且两者作用程度与中药提取液的浓度呈正相关。结论建立的sAA胶体金免疫渗滤法操作简便、快速,成本低,灵敏度和稳定性良好,可为sAA的快速检测提供新方法。Objective To develop a dot immunogold filtration assay for salivary alpha-amylase(sAA),prepare a test paper and apply to the research on enzymology of Atractylodes macrocephala Koidz and Pueraria lobata(Willd) Ohwi.Methods The pH value and amount of antibody to prepare colloidal gold nanoparticle-labeled antibody bioconjugates were optimized. A single factor experiment was designed to investigate eight factors,including type of coating solution,condition for coating,type of blocking buffer, condition for blocking, type of cleaning solution, optimal working concentrations of antigen and antibody,as well as reaction conditions for coating antigen with colloidal gold nanoparticlelabeled antibody and test samples with colloidal gold nanoparticle-labeled antibody,based on which a dot immunogold filtration assay was developed. The prepared test paper was verified for sensitivity and stability,and used for the promoting or inhibiting effects of extracts of Atractylodes macrocephala Koidz and Pueraria lobata(Willd)Ohwi,at various concentrations,on the specific binding activity of sAA to its antibody. Results The optimal pH value of colloidal gold for labeling of antibody was 8. 5,while the optimal dilution ratio of antibody was 1∶125. The colloidal gold nanoparticlelabeled antibody bioconjugates under the optimal condition showed good stability,of which the titer was 1∶384. The optimal condition for dot immunogold filtration assay was as follows :sAA was diluted with PBS buffer(0. 01 mol/L,pH 7. 4) containing 8% methanol,as a coating solution,to a concentration of 10 μg/mL,and incubated at 37 ℃ for 15 min for coating,then blocked with PBS buffer(0. 01 mol/L,pH 7. 4)containing 2% bovine serum albumin at 37 ℃ for 15 min. NC membrane was washed with PBS buffer(0. 01 mol/L,pH 7. 4) containing 0. 4% Tween-20. The test samples were reacted with the colloidal gold nanoparticle-labeled antibody at a dilution ratio of 1∶1 at 37 ℃ for 15 min then,after addition of NC membrane,at 25 ℃ for 20 min. The test result was

关 键 词：α-唾液淀粉酶 胶体金免疫渗滤法 中药 酶学作用 

分 类 号：R285.5[医药卫生—中药学]