Abstract
Key message
Plant expression platform is the new source of immunoglobulin G (IgG) toward small low-molecular-weight targets. The plant-made monoclonal antibody-based immunoassay exhibits comparable analytical performance with hybridoma antibody.
Abstract
Immunoassays for small molecules are efficiently applied for monitoring of serum therapeutic drug concentration, food toxins, environmental contamination, etc. Immunoglobulin G (IgG) is usually produced using hybridoma cells, which requires complicated procedures and expensive equipment. Plants can act as alternative and economic hosts for IgG production. However, the production of free hapten (low-molecular-weight target)-recognizing IgG from plants has not been successfully developed yet. The current study aimed at creating a plant platform as an affordable source of IgG for use in immunoassays and diagnostic tools. The functional IgG was expressed in Nicotiana benthamiana leaves infiltrated with Agrobacterium tumefaciens strain GV3101 with recombinant geminiviral vectors (pBY3R) occupying chimeric anti-miroestrol IgG genes. The appropriate assembly between heavy and light chains was achieved, and the yield of expression was 0.57 µg/g fresh N. benthamiana leaves. The binding characteristics of the IgG to miroestrol and binding specificity to related compounds, such as isomiroestrol and deoxymiroestrol, were similar to those of hybridoma-produced IgG (monoclonal antibody, mAb). The plant-based mAbs exhibited high sensitivity for miroestrol (IC50, 23.2 ± 2.1 ng/mL), precision (relative standard deviation ≤ 5.01%), and accuracy (97.8–103% recovery), as determined using quantitative enzyme-linked immunosorbent assay. The validated enzyme-linked immunosorbent assay was applicable to determine miroestrol in plant samples. Overall, the plant-produced functional IgG conserved the binding activity and specificity of the parent IgG derived from mammalian cells. Therefore, the plant expression system may be an efficient and affordable platform for the production of antibodies against low-molecular-weight targets in immunoassays.
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Acknowledgements
We also thank Professor Hugh Mason (Arizona State University) for providing the geminiviral vector.
Funding
The authors express their gratitude to the Faculty of Pharmaceutical Sciences, Chulalongkorn University, for providing a research fund (Grant number Phar2563-RG011) to WP and the Graduate School, Ratchadapisek Somphot Fund, for providing the financial support to KR. This research was financially supported by the new strategic research project (P2P), Walailak University, Thailand.
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KR, TK, AK, WS, AB, and GY conducted experiments. WP, SS, WP, and GY conceived and designed research. KR and GY wrote the manuscript. All authors read and approved the manuscript.
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Rattanapisit, K., Kitisripanya, T., Konyanee, A. et al. Plant-made antibody against miroestrol: a new platform for expression of full-length immunoglobulin G against small-molecule targets in immunoassays. Plant Cell Rep 40, 723–733 (2021). https://doi.org/10.1007/s00299-021-02670-z
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DOI: https://doi.org/10.1007/s00299-021-02670-z