New Approach for Development of Sensitive and Environmentally Friendly Immunoassay for Mycotoxin Fumonisin B1 Based on Using Peptide-MBP Fusion Protein as Substitute for Coating Antigen
New Approach for Development of Sensitive and Environmentally Friendly Immunoassay for Mycotoxin Fumonisin B1 Based on Using Peptide-MBP Fusion Protein as Substitute for Coating Antigen
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以肽-MBP融合蛋白替代包被抗原开发霉菌毒素伏马菌素B-1灵敏环保免疫分析新方法
DOI：
10.1021/ac502037w
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发表时间：
2014-08-19
影响因子：
7.4
通讯作者：
Xiong, Zheng-ping
中科院分区：
文献类型：
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作者：
Xu, Yang;Chen, Bo;Xiong, Zheng-ping
在此，我们以小分子分析物的模拟表位为基础，展示了一种基于多肽-MBP融合蛋白的、灵敏的、环境友好的有毒小分子分析物免疫分析的新方法。本工作以霉菌毒素伏马菌素B-1(FB1)为模型半抗原，从12肽文库中筛选出与抗FB1抗体结合的噬菌体展示肽(模拟表位)。将编码该序列的DNA克隆到大肠杆菌ER2738中，作为麦芽糖结合蛋白(MBP)的融合蛋白。所制备的多肽-MBP融合蛋白均一可克隆，不含FB1，可作为包被抗原用于免疫分析。用融合蛋白(F1-MBP和F15-MBP)建立的免疫定量检测方法的半数抑制浓度分别为2.15+/-0.13 ng/mL和1.26+/-0.08 ng/mL。利用融合蛋白F1-MBP建立了一种定性ELISPOT检测方法，其灵敏度比化学合成的FB1-BSA偶联物ELISPOT免疫检测方法高10倍。该多肽-MBP融合蛋白不仅可以大规模制备均一的、不含FB1的多肽-MBP融合蛋白，而且有助于建立一种分析FB1的高灵敏免疫分析方法。此外，这一新概念可能为各种有毒小分子的免疫分析提供一种通用方法的潜在应用。
Here, on the basis of mimotope of small analytes, we demonstrated a new approach for development of sensitive and environmentally friendly immunoassay for toxic small analytes based on the peptide-MBP fusion protein. In this work, using mycotoxin fumonisin B-1 (FB1) as a model hapten, phage displayed peptide (mimotope) that binds to the anti-FB1 antibody were selected by biopanning from a 12-mer peptide library. The DNA coding for the sequence of peptide was cloned into Escherichia coli ER2738 as a fusion protein with a maltose binding protein (MBP). The prepared peptide-MBP fusion protein are "clonable" homogeneous and FB1-free products and can be used as a coating antigen in the immunoassay. The half inhibition concentration of the quantitative immunoassay setup with fusion protein (F1-MBP and F15-MBP) was 2.15 +/- 0.13 ng/mL and 1.26 +/- 0.08 ng/mL, respectively. The fusion protein (F1-MBP) was also used to develop a qualitative Elispot assay with a cutoff level of 2.5 ng/mL, which was 10-fold more sensitive than that measured for chemically synthesized FB1-BSA conjugates based Elispot immunoassay. The peptide-MBP fusion protein not only can be prepared reproducibly as homogeneous and FB1-free products in a large-scale but also can contribute to the development of a highly sensitive immunoassay for analyzing FB1. Furthermore, the novel concept might provide potential applications to a general method for the immunoassay of various toxic small molecules.