One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein.
One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein.
复制标题
DOI：
10.1021/ac504735p
复制
发表时间：
2015
影响因子：
7.4
通讯作者：
Hammock, Bruce D.
中科院分区：
文献类型：
--
作者：
Wang, Jia;Majkova, Zuzana;Bever, Candace R. S.;Yang, Jun;Gee, Shirley J.;Li, Ji;Xu, Ting;Hammock, Bruce D.
四溴双酚A （TBBPA）是一种普遍存在的溴化阻燃剂，广泛存在于环境和人体中。重链抗体（VHH）的可变结构域，天然存在于骆驼中，接近功能性抗原结合实体的下限。易于基因操作使得这种vhs作为免疫试剂使用的优越选择。本研究表达了与大肠杆菌碱性磷酸酶（AP）融合的高选择性抗tbbpa VHH T3-15，显示了tbbpa的综合结合能力和酶活性。在5%二甲亚砜(DMSO)/磷酸盐缓冲盐水（PBS, pH 7.4）中，建立了基于融合蛋白T3-15-AP的一步免疫分析TBBPA，一半最大信号抑制浓度（IC50）为0.20 ng mL−1。与亲本VHH T3-15相比，T3-15- ap能够结合更多种类的包衣抗原，检测灵敏度略有提高。T3-15-AP与一组重要的溴化类似物的交叉反应性可忽略不计（<0.1%）。虽然T3-15-AP易受极端高温（90°C）的影响，但在环境温度下，基于T3-15-AP的实验中观察到至少70天的结合稳定性要高得多。建立了一种用DMSO稀释尿液样品的简单预处理方法，用于一步测定。一步法测定尿液中TBBPA的回收率为96.7 ~ 109.9%，与HPLC-MS/MS法具有良好的相关性。该二聚体融合蛋白VHH-AP在人体暴露和环境监测方面具有广阔的应用前景。
Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environment and human exposures. A variable domain of the heavy chain antibody (VHH), naturally occurring in camelids, approaches the lower size limit of functional antigen-binding entities. Ease of genetic manipulation makes such VHHs a superior choice to use as an immunoreagent. In this study, a highly selective anti-TBBPA VHH T3-15 fused with alkaline phosphatase (AP) from E. coli was expressed, showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on the fusion protein T3-15-AP was developed for TBBPA in 5% dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH 7.4), with a half-maximum signal inhibition concentration (IC50) of 0.20 ng mL−1. Compared to the parental VHH T3-15, T3-15-AP was able to bind to a wider variety of coating antigens and the assay sensitivity was slightly improved. Cross-reactivity of T3-15-AP with a set of important brominated analogs was negligible (<0.1%). Although T3-15-AP was susceptible to extreme heat (90 °C), much higher binding stability at ambient temperature was observed in the T3-15-AP based assay for at least 70 days. A simple pretreatment method of diluting urine samples with DMSO was developed for a one-step assay. The recoveries of TBBPA from urine samples by this one-step assay ranged from 96.7–109.9% and correlated well with an HPLC-MS/MS method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring.
登录
查看更多内容
影响因子：
7.4
作者：
Bever, Candace R. S.;Majkova, Zuzana;Radhakrishnan, Rajeswaran;Suni, Ian;McCoy, Mark;Wang, Yanru;Dechant, Julie;Gee, Shirley;Hammock, Bruce D.
通讯作者：
Hammock, Bruce D.
影响因子：
7.4
作者：
Kim, Hee-Joo;McCoy, Mark R.;Majkova, Zuzana;Dechant, Julie E.;Gee, Shirley J.;Tabares-da Rosa, Sofia;Gonzalez-Sapienza, Gualberto G.;Hammock, Bruce D.
通讯作者：
Hammock, Bruce D.
影响因子：
48
作者：
Rothbauer, Ulrich;Zolghadr, Kourosh;Leonhardt, Heinrich
通讯作者：
Leonhardt, Heinrich
影响因子：
8
作者：
Dumoulin, M;Conrath, K;Matagne, A
通讯作者：
Matagne, A
影响因子：
2.8
作者：
Ismaili, Ahmad;Jalali-Javaran, Mokhtar;Memari, Hamid Raiabi
通讯作者：
Memari, Hamid Raiabi