Citation: CAO Dong-Mei, XU Yang, TU Zhui, LI Yan-Ping, XIONG Liang, FU Jin-Heng. One-step Enzyme Linked Immunosorbent Assay for Detection of Aflatoxin B₁ Using a Nanobody-Alkaline Phosphatase Fusion Protein[J]. Chinese Journal of Analytical Chemistry, ;2016, 44(7): 1085-1091. doi: 10.11895/j.issn.0253-3820.150902 shu

One-step Enzyme Linked Immunosorbent Assay for Detection of Aflatoxin B₁ Using a Nanobody-Alkaline Phosphatase Fusion Protein

CAO Dong-Mei ^1, ,
XU Yang ^1,2 ,
TU Zhui ¹ ,
LI Yan-Ping ² ,
XIONG Liang ^1,3 ,
FU Jin-Heng ²

1.
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China;
2.
Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, China;
3.
Department of Preventive Medicine, Gannan Medical College, Ganzhou 341000, China

Received Date: 13 November 2015
Revised Date: 8 April 2016

Fund Project: This work was supported by the National Natural Science Foundation of China (No. 31301479), the Research Foundation for Young Scientists of State Key Laboratory of Food Science and Technology (No. SKLF-QN-201513), and the Major Program of Natural Science Foundation of Jiangxi Province (20152ACB20005)

Figures(5)

A phage clone (designated as the anti-aflatoxin B₁ nanobody, G8) specific binding to aflatoxin B₁ (AFB₁) was screened from an immunized nanobody phage library. The DNA fragment encoding G8 was subcloned into the vector pET25b(+)-alkaline phosphatase (AP), and fused to the N terminal of AP. The fusion protein G8-AP was expressed in E. coli BL21 (Rosetta, DE3) as soluble protein, and purified by immobilized metal affinity chromatography. The purified G8-AP exhibited AP activity of 364.5±8.3 U/mg. Enzyme-linked immunosorbent assay (ELISA) showed that G8-AP was also capable of recognizing AFB₁, with negligible cross-reactivity toward other six common mycotoxins. A one-step ELISA was established to detect AFB₁ using G8-AP. The effects of methanol concentration, ionic strength, and pH on the detection sensitivity of the G8-AP-ELISA were evaluated and optimized. Under the optimized conditions (methanol 20%, NaCl 20 mmol/L, pH 7.4), the half inhibitory concentration (IC₅₀) of the G8-AP based ELISA was 19.8 ng/mL, with the linear range of 4.3-92 ng/mL and the detection limit of 2.6 ng/mL. The recoveries of AFB₁ for corn and wheat samples were 90.4%-101.5%.
- Anti-aflatoxin B₁ Nanobody,
- Alkaline phosphatase,
- Fusion expression,
- Enzyme-linked immunosorbent assay
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沈阳化工大学材料科学与工程学院沈阳 110142

One-step Enzyme Linked Immunosorbent Assay for Detection of Aflatoxin B1 Using a Nanobody-Alkaline Phosphatase Fusion Protein

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通讯作者: 陈斌, bchen63@163.com

One-step Enzyme Linked Immunosorbent Assay for Detection of Aflatoxin B₁ Using a Nanobody-Alkaline Phosphatase Fusion Protein