CAJ | 학술논문

提出了一种简便、高灵敏的荧光免疫传感新技术,通过抗体/抗原/核酸适配体-质粒DNA复合物的特异性识别与双链质粒DNA与荧光染料SYBR Green Ⅰ的嵌合作用,实现对血小板衍生增长因子BB(PDGF-BB)的检测.生物识别反应在微孔板中进行,PDGF-BB抗原与微孔板底部预包被的PDGF-BB抗体免疫反应后,加入核酸适配体-质粒DNA复合物与抗原形成夹心复合物.加入DNA双链嵌合染料SYBR Green Ⅰ与夹心复合物的双链DNA部分结合可产生强荧光,其荧光强度可用于定量测定PDGF-BB浓度.实验考察了离子浓度、核酸适配体的延伸引物片段与质粒PUC19的反应比例、染料SYBR Green Ⅰ浓度等分析条件对荧光信号的影响.在优化反应条件下,PDGF-BB检测的线性范围为0.2～200 μg/L,检出限为0.1 μg/L,并且实现了对人血清中PDGF-BB的定量检测.
제출료일충간편、고령민적형광면역전감신기술,통과항체/항원/핵산괄배체-질립DNA복합물적특이성식별여쌍련질립DNA여형광염료SYBR Green Ⅰ적감합작용,실현대혈소판연생증장인자BB(PDGF-BB)적검측.생물식별반응재미공판중진행,PDGF-BB항원여미공판저부예포피적PDGF-BB항체면역반응후,가입핵산괄배체-질립DNA복합물여항원형성협심복합물.가입DNA쌍련감합염료SYBR Green Ⅰ여협심복합물적쌍련DNA부분결합가산생강형광,기형광강도가용우정량측정PDGF-BB농도.실험고찰료리자농도、핵산괄배체적연신인물편단여질립PUC19적반응비례、염료SYBR Green Ⅰ농도등분석조건대형광신호적영향.재우화반응조건하,PDGF-BB검측적선성범위위0.2～200 μg/L,검출한위0.1 μg/L,병차실현료대인혈청중PDGF-BB적정량검측.
A novel simple,sensitive fluorescence immunosensing method based on aptamer-plasmid complex amplification was developed. This method utilized the specific recognition between antibody and antigen as well as aptamer-plasmid complex and the intercalation of fluorescence dye SYBR Green Ⅰ in the groove of duplex plasmid DNA in detection of Platelet-Derived Growth Factor BB (PDGF-BB). The immunoassay was performed in the microtiter wells in which rabbit anti PDGF-BB antibody was immobilized. The PDGF BB analyte was captured by the primary antibody and then sandwiched by the aptamer-plasmid DNA complex. The introduction of fluorescence dye SYBR Green Ⅰ allows for the detection of the sandwiched immunocomplex of antibody/anigen/aptamer-plasmid complex. Under the optimized conditions of salt concentration,ratio of aptamer to PUC19,and SYBR Green Ⅰ concentration,the proposed method offers a linear detection range from 0.2 μg/L to 200 μg/L with a detection limit of 0.1μg/L.