Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies.
Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies.
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DOI：
10.1021/acs.analchem.7b03190
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发表时间：
2017-10-17
影响因子：
7.4
通讯作者：
Hammock BD
中科院分区：
文献类型：
--
作者：
Cui X;Vasylieva N;Wu P;Barnych B;Yang J;Shen D;He Q;Gee SJ;Zhao S;Hammock BD
甘氨胆酸（GCA）是胆汁酸的重要代谢产物，其尿液水平有望成为肝细胞癌（HCC）的特异性诊断生物标志物。GCA的高通量免疫检测方法对肝癌的早期诊断、监测和早期发现具有重要意义。单链可变片段（scFv）抗体具有几个期望的特性，并且是用于免疫测定的传统抗体的有吸引力的替代物。由于鸡抗体具有单一的重链和轻链可变功能结构域，因此它是用于简化产生用于GCA检测的重组抗体的理想框架。然而，鸡scFv很少用于检测GCA。本研究以GCA半抗原与牛血清白蛋白（BSA）偶联免疫鸡，构建了单链抗体库，并通过噬菌体展示技术分离了抗GCA的单链抗体。与同源包被抗原相比，使用异源包被抗原导致免疫测定的灵敏度提高约85倍。在优化条件下，该方法的线性范围为0.02 ~ 0.18 μg/mL，IC 50为0.06 μg/mL。除牛磺胆酸外，该试验显示与各种相关胆汁酸的交叉反应性可忽略不计。加标人尿样中GCA的检出率范围为86.7%至123.3%。这些结果结合scFv抗体的优点，表明基于鸡scFv的ELISA是用于高通量筛选人尿液中GCA的合适方法。
Glycocholic acid (GCA) is an important metabolite of bile acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, it is an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, the chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, using a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity in the immunoassay. This assay, under optimized conditions, had a linear range of 0.02 ~ 0.18 μg/mL, with an IC50 of 0.06 μg/mL. The assay showed negligible cross-reactivity with various related bile acids, except for taurocholic acid. The detection of GCA from spiked human urine samples ranged from 86.7 to 123.3 %. These results, combined with the advantage of the scFv antibodies, indicated that a chicken scFv-based ELISA is a suitable method for high-throughput screening of GCA in human urine.
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影响因子：
29.4
作者：
El-Serag HB
通讯作者：
El-Serag HB
影响因子：
2.9
作者：
Mittal S;El-Serag HB
通讯作者：
El-Serag HB
影响因子：
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作者：
Ahmad ZA;Yeap SK;Ali AM;Ho WY;Alitheen NB;Hamid M
通讯作者：
Hamid M
影响因子：
2.5
作者：
Lipman, NS;Jackson, LR;Weis-Garcia, F
通讯作者：
Weis-Garcia, F
影响因子：
2.7
作者：
Kobayashi, N;Katsumata, H;Mizuuchi, Y
通讯作者：
Mizuuchi, Y