基于胶体金的8-羟基-2'-脱氧鸟苷免疫层析试纸条

Development of a colloidal gold based immunochromatographic strip for 8-OHdG detection

摘要:

8-羟基-2'-脱氧鸟苷(8-hydroxy-2'-deoxyguanosine,8-OHdG)是评价DNA氧化损伤较灵敏和稳定的生物标志物。文中采用竞争法建立一种快速、灵敏检测8-OHdG的胶体金免疫层析试纸条。将样品垫(玻璃纤维素膜)、结合垫(玻璃纤维素膜)、硝酸纤维素膜和吸水垫依此粘贴在聚氯乙烯(polyvinyl chloride,PVC)底板上，构建试纸条。通过柠檬酸钠还原三水合四氯金酸制备胶体金(gold nanoparticles,AuNPs)，8-OHdG配对的抗体(antibody,Ab)包被于AuNPs的外层(Ab coated AuNPs,Ab@AuNPs)作为探针。牛血清蛋白(bovine serum protein,BSA)与8-OHdG用碳二亚胺盐酸盐偶联制备人工抗原，作为检测线的包被抗原。羊抗鼠多抗(imunoglobulin G,IgG)作为质控线的包被抗体。对试纸条的硝酸纤维素膜、上样液的配方、金标抗体喷涂量等实验参数进行了优化。结果表明，硝酸纤维素膜(nitrocellulose film,NC)膜采用CN 95，上样液的最优配方为1% BSA+3%吐温-20+3%蔗糖+0.9% NaCl溶液，最适金标抗体喷涂量为4 μL。利用试纸条在可见光下检测8-OHdG，根据检测线(test line,T线)和质控线(control line,C线)的显色强度对比，可初步判断尿液中8-OHdG的含量水平。并通过T线的灰度值计算尿液中的8-OHdG的浓度，检测限为2.55 μg/L。该方法简单、快速且有较好的特异性，可检测人体尿液中的8-OHdG含量，以初步评价人体的健康状态。

Abstract:

8-hydroxy-2'-deoxyguanosine (8-OHdG) is a sensitive and stable biomarker for evaluating DNA oxidative damage.A rapid and sensitive colloidal gold immunochromatographic strip was developed for 8-OHdG detection by a competitive method.The sample pad (glass cellulose film),bonding pad (glass cellulose film),nitrocellulose film and absorbent pad were pasted on the polyvinyl chloride (PVC) base plate to construct the test strip.Colloidal gold (AuNPs) was prepared by the reduction of chloroauric acid with sodium citrate.8-OHdG antibody (Ab) was coated on the outer layer of AuNPs to form Ab@AuNPs as a probe.Bovine serum albumin (BSA) and 8-OHdG were conjugated with carbodiimide hydrochloride to prepare an artificial antigen,which was used as the coating antigen of detection line.Goat anti mouse polyclonal antibody IgG was used as the coating antibody of control line.The experimental parameters were optimized including the type of nitrocellulose membrane,the formula of loading solution,and the spraying amount of gold labeled antibody.The results showed that the appropriate nitrocellulose membrane was CN 95.The optimal loading solution included BSA (1%),Tween-20(3%),sucrose (3%) and NaCl (0.9%).The optimal spraying amount of gold labeled antibody was 4 μL.8-OHdG can be detected by the strip under visible light,and the level of 8-OHdG in urine can be preliminarily determined by comparing the color intensity of T line and C line.The 8-OHdG concentration in urine was further calculated by the gray value of T line and the threshold of detection was 2.55 μg/L.This colloidal gold immunochromatographic strip is simple,rapid and specific for detecting 8-OHdG in human urine to preliminarily evaluate the human status.