荧光免疫层析法定量检测胃蛋白酶原及应用

目的 建立一种一次操作即可同时定量检测胃蛋白酶原Ⅰ及胃蛋白酶原Ⅱ荧光免疫层析的方法,检测人血清中胃蛋白酶原Ⅰ(PGⅠ)及胃蛋白酶原Ⅱ(PGⅡ)的含量,用于辅助诊断慢性萎缩性胃炎和早期胃癌.方法 将鼠抗胃蛋白酶原Ⅰ (PGⅠ)抗体用划线机喷到硝酸纤维素膜表面作为检测线1(T1),将鼠抗胃蛋白酶原Ⅱ(PGⅡ)抗体用划线机喷到硝酸纤维素膜表面作为检测线2(T2),将羊抗兔IgG多克隆抗体喷到硝酸纤维素膜表面作为质控线(C);用荧光素分别标记鼠抗胃蛋白酶原Ⅰ(PGⅠ)抗体、鼠抗胃蛋白酶原Ⅱ(PGⅡ)抗体和兔IgG抗体,将所制得的以上三种抗体荧光素标记物按一定比例混合,均匀喷到玻璃纤维素膜上制得荧光标记物垫;以双抗体夹心法反应模式制备同时定量检测胃蛋白酶原Ⅰ及胃蛋白酶原Ⅱ的荧光免疫层析法试纸条,并使用酶联免疫法试剂进行比对,对方法的线性、精密度、最低检出限、回收率、相关性等方面进行分析.结果 荧光免疫层析法检测胃蛋白酶原Ⅰ及胃蛋白酶原Ⅱ,进行Logistic曲线四参数拟合,R2均为0.99,拟合度较高;精密性CV分别为3.59％、3.40％;荧光免疫层析法与酶联免疫法的相关性较好(PG Ⅰ和PGⅡ分别为r=0.99,r =0.99);最低检出限分别为0.81 ng/mL和0.72ng/mL.结论 所建立的在一个检测条上同时测定胃蛋白酶原Ⅰ(PGⅠ)及胃蛋白酶原Ⅱ(PGⅡ)含量的荧光免疫层析法方法,具有较好的特异性和较高的灵敏度,PG Ⅰ和PGⅡ无相互干扰,结果准确、操作便捷,便于我国基层疾控中心及医疗结构对胃蛋白酶原的检测.

Development of Pepsinogen Immunity Chromatography Assay Using Fluorescence Lanthanide Nanoparticles

Objective To develop a lateral flow immunechromatography assay using fluorescence to detect the quantity of pepsinogen Ⅰ and pepsinogenⅡ in serum respectively,which can be used in the diagnosis of early gastric cancer.Methods A lateral-flow(LF) device using the luminescent lanthanide nanoparticles as reporter was applied to detect the concentration of pepsinogen Ⅰ and pepsinogen Ⅱ respectively at the same time in one test.To make this device,anti-pepsinogen Ⅰ (PG Ⅰ) antibodies,anti-pepsinogen Ⅱ (PG Ⅱ) antibodies and goat anti-rabbit IgG were coated on the nitrocellulose membrane respectively,fluorescent labeled antibodies to pepsinogen Ⅰ (PG Ⅰ),fluorescent labeled antibodies to pepsinogen Ⅱ (PG Ⅱ) and fluorescent labeled rabbit IgG were coated on the glass fiber mixed.The performance of the device such as detection limit,precision and specificity was evaluated compared with enzyme-linked immunoassay reagent.Results Using the method as reference for detection of PG Ⅰ,the correlation coefficient (R),lower limit of detection(LLD),coefficient of variation (CV) and correlation compared with ELISA (R) were 0.99,0.81ng/mL,8.17％ and 0.99 respectively.And those were 0.99,0.72ng/mL,4.66％ and 0.99 while detection of PGⅡ.Conclusion A fluorescence immunechromatography assay for the quantitative detection of pepsinogen Ⅰ and pepsinogen Ⅱ in serum was developed.These assays were non-invasive,simple and economic test and could be used in early gastric cancer screening suitably.