Production and Characterization of a Single-Chain Variable Fragment Linked Alkaline Phosphatase Fusion Protein for Detection ofO,O-Diethyl Organophosphorus Pesticides in a One-Step Enzyme-Linked Immunosorbent Assay

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL−1 and a limit of detection of 0.059 ng mL−1, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice.

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A one-step chemiluminescence immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single chain Fv–alkaline phosphatase fusion protein

Analytical Methods ◽

10.1039/c5ay01410g ◽

2015 ◽

Vol 7 (21) ◽

pp. 9032-9039 ◽

Cited By ~ 13

Author(s):

Xuezhi Yu ◽

Xiaoqi Tao ◽

Jianzhong Shen ◽

Suxia Zhang ◽

Xingyuan Cao ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Fusion Protein ◽

Immunosorbent Assay ◽

Single Chain ◽

Chemiluminescence Immunoassay ◽

Single Chain Fv ◽

One Step

A simple and rapid chemiluminescence competitive direct enzyme-linked generic immunosorbent assay was developed for 20 FQs in fish and shrimp samples.

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Bacterial Expression of a Single-Chain Variable Fragment (scFv) Antibody against Ganoderic Acid A: A Cost-Effective Approach for Quantitative Analysis Using the scFv-Based Enzyme-Linked Immunosorbent Assay

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.b17-00531 ◽

2017 ◽

Vol 40 (10) ◽

pp. 1767-1774 ◽

Cited By ~ 6

Author(s):

Gorawit Yusakul ◽

Poomraphie Nuntawong ◽

Seiichi Sakamoto ◽

Pahweenvaj Ratnatilaka Na Bhuket ◽

Toshitaka Kohno ◽

...

Keyword(s):

Quantitative Analysis ◽

Immunosorbent Assay ◽

Cost Effective ◽

Bacterial Expression ◽

Scfv Antibody ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Ganoderic Acid ◽

Cost Effective Approach

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Construction of Single Chain Variable Fragment (ScFv) and BiscFv-Alkaline Phosphatase Fusion Protein for Detection ofBacillusAnthracis

Analytical Chemistry ◽

10.1021/ac0512352 ◽

2006 ◽

Vol 78 (4) ◽

pp. 997-1004 ◽

Cited By ~ 57

Author(s):

Shi-Hua Wang ◽

Ji-Bin Zhang ◽

Zhi-Ping Zhang ◽

Ya-Feng Zhou ◽

Rui-Fu Yang ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Fusion Protein ◽

Single Chain Variable Fragment ◽

Single Chain

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Construction of a single-chain variable fragment antibody against daidzin and its potential use in an enzyme-linked immunosorbent assay

Planta Medica ◽

10.1055/s-0033-1352424 ◽

2013 ◽

Vol 79 (13) ◽

Author(s):

B Pongkitwitoon ◽

H Tanaka ◽

K Tabata ◽

S Morimoto

Keyword(s):

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Potential Use

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Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/cjm-2014-0468 ◽

2014 ◽

Vol 60 (11) ◽

pp. 737-743 ◽

Cited By ~ 3

Author(s):

Weifeng Chen ◽

Li Hu ◽

Aiping Liu ◽

Jinquan Li ◽

Fusheng Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Food Poisoning ◽

Staphylococcal Enterotoxin ◽

Enzyme Linked Immunosorbent Assay ◽

Staphylococcal Enterotoxin A ◽

Single Chain Variable Fragment ◽

Single Chain

The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10−8 mol·L−1, its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10−7 mol·L−1. The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L−1.

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