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      Development of a sensitive monoclonal antibody-based sandwich ELISA to detect Vip3Aa in genetically modified crops

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          Abstract

          Objectives

          To develop a sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) to detect Vip3Aa in genetically modified (GM) crops and their products.

          Results

          Vegetative insecticidal proteins (Vips) are secreted by Bacillus thuringiensis ( Bt) and are known to be toxic to Lepidoptera species. Vip3Aa family proteins, Vip3Aa19 and Vip3Aa20, were successfully applied in GM crops to confer an effective and persistent insecticidal resistance. A sensitive monoclonal antibody-based sandwich ELISA was developed to detect Vip3Aa in GM crops and their products. Two monoclonal antibodies were raised against the overexpressed and purified His-Vip3Aa20, were purified from mouse ascites and characterized. A sandwich ELISA method was developed using the 2G3-1D7 monoclonal antibody for capture and the biotin-labeled 1F9-1F5 monoclonal antibody for detection of Vip3Aa20. The linear detection range of the method was found to be approximately 31.25–500 pg/ml, with a sensitivity of 10.24 pg/ml.

          Conclusions

          The established ELISA was effective for detecting Vip3Aa family proteins other than Vip3Aa8, and was successfully applied in the detection of Vip3Aa20 and Vip3Aa19 expressed in transgenic maize and cotton.

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          Most cited references36

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          Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.

          Franklin Mullins, J J Estruch, Kenneth G Warren … (1996)
          A novel vegetative insecticidal gene, vip3A(a), whose gene product shows activity against lepidopteran insect larvae including black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), tobacco budworm (Heliothis virescens), and corn earworm (Helicoverpa zea) has been isolated from Bacillus thuringiensis strain AB88. VIP3-insecticidal gene homologues have been detected in approximately 15% of Bacillus strains analyzed. The sequence of the vip3A(b) gene, a homologue of vip3A(a) isolated from B. thuringiensis strain AB424 is also reported. Vip3A(a) and (b) proteins confer upon Escherichia coli insecticidal activity against the lepidopteran insect larvae mentioned above. The sequence of the gene predicts a 791-amino acid (88.5 kDa) protein that contains no homology with known proteins. Vip3A insecticidal proteins are secreted without N-terminal processing. Unlike the B. thuringiensis 5-endotoxins, whose expression is restricted to sporulation, Vip3A insecticidal proteins are expressed in the vegetative stage of growth starting at mid-log phase as well as during sporulation. Vip3A represents a novel class of proteins insecticidal to lepidopteran insect larvae.
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            Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay.

            J.David Beatty, Barbara Beatty, William Vlahos (1987)
            Enzyme-linked immunoadsorbent assay (EIA) has widespread use for the measurement of antibody concentration. The affinity constant (Kaff) of the antibody has an effect upon the quantification by EIA. It is thus important to be able to measure Kaff by solid-phase EIA. Based upon the Law of Mass Action and using serial dilutions of both antigens (coating the plate) and antibody, Kaff has been measured by EIA. A microtiter plate was coated with antigen (Ag) and then incubated with monoclonal antibody (Ab). The plate was sequentially incubated with a second enzyme-antibody conjugate (EAC) and with the enzyme substrate. The amount of Ab adherent to Ag on the plate [Ag Ab] and [Ag2 Ab] was reflected by the enzyme product measured by OD. The use of serial dilutions of Ab resulted in a sigmoid curve of OD versus logarithm of total Ab added to the well. Comparison of the OD at the upper plateau (OD-100) for different antibodies was a reflection of the relative number of epitopes on the Ag that were identified by the different antibodies, provided excessive EAC was used. [Ab]t and [Ab']t were the measurable total antibody concentrations in the wells at OD-50 and OD-50' for plates coated with [Ag] and [Ag'], respectively. [Ag] and [Ag'] were not true antigen concentrations, but were a measure of antigen density on the plate. For [Ag'] = [Ag]/2, Kaff = 1/2(2[Ab']t-[Ab]t. Using five different anti-CEA antibodies and different proportions of CEA in the coating solution, Kaff was measured. Kaff determined by EIA correlated well with Kaff measured by soluble phase inhibition assay. This EIA method of estimation of Kaff is simple, rapid, and reliable.
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              Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria.

              Colin Berry, H Ernest Schnepf, Neil Crickmore … (2002)
              Gram-positive spore-forming entomopathogenic bacteria can utilize a large variety of protein toxins to help them invade, infect, and finally kill their hosts, through their action on the insect midgut. These toxins belong to a number of homology groups containing a diversity of protein structures and modes of action. In many cases, the toxins consist of unique folds or novel combinations of domains having known protein folds. Some of the toxins display a similar structure and mode of action to certain toxins of mammalian pathogens, suggesting a common evolutionary origin. Most of these toxins are produced in large amounts during sporulation and have the remarkable feature that they are localized in parasporal crystals. Localization of multiple toxin-encoding genes on plasmids together with mobilizable elements enables bacteria to shuffle their armory of toxins. Recombination between toxin genes and sequence divergence has resulted in a wide range of host specificities.
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                Author and article information

                Contributors
                Weixiao Liu: liuweixiao@caas.cn
                Wujun Jin: jinwujun@caas.cn
                Journal
                Journal ID (nlm-ta): Biotechnol Lett
                Journal ID (iso-abbrev): Biotechnol. Lett
                Title: Biotechnology Letters
                Publisher: Springer Netherlands (Dordrecht )
                ISSN (Print): 0141-5492
                ISSN (Electronic): 1573-6776
                Publication date (Electronic): 5 March 2020
                Publication date PMC-release: 5 March 2020
                Publication date (Print): 2020
                Volume: 42
                Issue: 8
                Pages: 1467-1478
                Affiliations
                [1 ]GRID grid.410727.7, ISNI 0000 0001 0526 1937, Biotechnology Research Institute, , Chinese Academy of Agricultural Sciences, ; Beijing, 100081 China
                [2 ]Department of Food and Biological Engineering, Handan Polytechnic College, Handan, 056001 China
                [3 ]GRID grid.9227.e, ISNI 0000000119573309, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, , Chinese Academy of Science, ; Beijing, 100101 China
                Article
                Publisher ID: 2854
                DOI: 10.1007/s10529-020-02854-9
                PMC ID: 7354279
                PubMed ID: 32140882
                SO-VID: bad9e365-8c3b-40f2-b5aa-5b3e753a68c3
                Copyright © © The Author(s) 2020
                License:

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                Date received : 17 September 2019
                Date accepted : 27 February 2020
                Funding
                Funded by: Fundamental Research Funds for Non-profit Scientific Institution
                Award ID: Grant no. 1610392018008
                Award Recipient :
                Funded by: National Transgenic Major Program of China
                Award ID: No. 2017ZX08013001
                Award Recipient :
                Categories
                Subject: Original Research Paper
                Custom metadata
                issue-copyright-statement © Springer Nature B.V. 2020

                ScienceOpen disciplines: Biotechnology
                Keywords: vip3aa,monoclonal antibody,elisa,genetically modified crops,cotton,maize
                Data availability:
                ScienceOpen disciplines: Biotechnology
                Keywords: vip3aa, monoclonal antibody, elisa, genetically modified crops, cotton, maize

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