One-step immunoassay for the insecticide carbaryl using a chicken single-chain variable fragment (scFv) fused to alkaline phosphatase
Introduction
The insecticide carbaryl (1-naphthyl methylcarbamate) has been applied to over 120 different crops for insect control due to its inhibitory effect on acetylcholinesterase [1]. Carbaryl remains the third-most-used insecticide in the United States for home gardens, commercial agriculture, forestry and rangeland protection [2]. The widespread use of carbaryl has caused environmental pollution and food contamination [2]. Some adverse effects such as alteration of liver microsomal enzymes [3] and subacute neurotoxicity [4] have been observed after long-term exposure to carbaryl. The maximum residue limits (MRL) of carbaryl in some food resources were issued in different countries. For example, the United States Environmental Protection Agency sets carbaryl MRL at 5 and 0.1 mg kg−1 for vegetables, nuts and meat, respectively, while the Ministry of Agriculture and Rural Affairs of China sets carbaryl MRL at 1.0 mg kg−1 in vegetables, fruits, grains and oils. The increasing concern about the presence of carbaryl residues in the environment and food matrices has been driving a search for high-throughput detection methods for this pesticide.
Enzyme-linked immunosorbent assays (ELISAs) have shown to be a rapid and effective method for monitoring carbaryl in a variety of matrices [[5], [6], [7], [8], [9]]. Conventional polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs) have been commonly employed for the development of immunoassays, but the production of pAbs has the inevitable limitations in terms of its standardization and reproducibility while generation of mAbs requires lengthy process. In contrast, recombinant antibodies such as single-chain variable fragment antibody (scFv) can be easily stored, transformed, manipulated and expressed in bacteria [10,11] and thus, have been generated to develop immunoassays for the detection of pesticides (e.g. carbaryl) [12].
Unlike mammals’ immunoglobulin germline, only one functional variable heavy and light chain (VH and VL) gene segment exists in chickens and the antibody diversity is preserved by gene rearrangement and recombination. This peculiar mechanism of immunoglobulin gene diversification leads to only one set of primers being required for each antibody chain, instead of the mixtures needed for amplification of the variable gene families from other animals. Thus, constructing a phage library is technically easier using chickens compared with other animals. The generation and usage of chicken scFv are of a predominant interest in the area of diagnosis and therapy. For instance, chicken scFvs against snake venom [13], infectious bursal disease virus [14], staphylococcus aureus [15], and infectious bronchitis virus [16] have been generated for therapeutic purposes. Advances of recombinant DNA technology make it easy to genetically construct chicken scFv and alkaline phosphatase fusion proteins (scFv-AP), which have been applied in direct immunoassays for pathogenic bacteria to simplify the process [17,18].
Chicken scFvs have recently been used to detect small molecules in different matrices, such as glycocholic acid in human urine [19] and gentamycin in animal-derived food [20], but the usage in pesticide detection is scarcely. The specific objective of this study was to extend the application of chicken scFvs to pesticide detection in the environment and foods. Two anti-carbaryl scFvs were generated and genetically fused with AP to develop a rapid one-step ELISA for the detection of carbaryl in soil, apple and pear samples. Meanwhile, the binding ability of the scFv-AP fusion proteins to carbaryl was measured and compared with the parental scFvs. To the best of our knowledge, this is the first work to use chicken scFv as an effective immunoreagent for pesticide analysis in the environment.
Section snippets
Safety
Carbamate pesticides were discarded as hazardous wastes. All animal experimental protocols were reviewed and approved by the Ethics Committee of China Agricultural University for the Use of Laboratory Animals. Information about materials, chemicals and reagents is detailed in the supplementary data.
Animal immunization
Haptens C1
C6 (Fig. 1) were synthesized and coupled to the carrier proteins according to the previous reports [5,6]. Hapten C1 conjugated to keyhole limpet hemocyanin (KLH) was used for animal
Selection of the carbaryl hapten for immunization
In the generation of antibodies specific for small molecules, it is generally accepted that a suitable hapten for immunization should preserve the chemical and physical properties of the target compound. Carbaryl is susceptible to the chemical hydrolysis of the carbamate functional group, producing 1-naphthol [21]. Hence, haptens preserving the carbamate group are not promising for the generation of antibodies against carbaryl and several stable heterologous haptens showing some similarities to
Conclusions
The current study demonstrated a novel one-step ELISA for the rapid detection of carbaryl in soil and fruit samples using a chicken scFv-AP fusion protein. Two chicken-derived scFvs X1 and X2 with high affinity to carbaryl were isolated from a carbaryl-immunized library and genetically fused with AP, exhibiting an integrated carbaryl-binding capacity and enzymatic activity. The fusion of scFv and AP increased the binding activity of the scFv as the scFv-AP-based assays exhibited greater
Author contribution statement
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Acknowledgements
This work was supported in part by the Key Project of Intergovernmental International Scientific and Technological Innovation Cooperation, 2016YFE0108900, the National Key Research and Development Program of China, 2016YFD0800606, and the National Institute of Environmental Health Sciences Superfund Research Program, P42ES04699, USA. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
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