Abstract
Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 107 CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized. Among them, clone scFv-3H9 (MG214869) showing relative stable and high binding abilities to six Cry1 toxins was selected for expression and purification. SDS-PAGE indicated that the scFv-3H9 fragments approximately 27 kDa were successfully expressed in Escherichia coli HB2151 strain. The purified scFv-3H9 was used to establish the double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) for detecting six Cry1 toxins, of which the lowest detectable limits (LOD) and the lowest quantitative limits (LOQ) were 3.14–11.07 and 8.22–39.44 ng mL−1, respectively, with the correlation coefficient higher than 0.997. The average recoveries of Cry1 toxins from spiked rice leaf samples were ranged from 84 to 95%, with coefficient of variation (CV) less than 8.2%, showing good accuracy for the multi-residue determination of six Cry1 toxins in agricultural samples. This research suggested that the constructed phage display antibody library based on the animal which was immunized with the mixture of several antigens under the same category can be used for the quick and effective screening of generic antibodies.
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References
Abi-Ghanem D, Waghela SD, Caldwell DJ, Danforth HD, Berghman LR (2008) Phage display selection and characterization of single-chain recombinant antibodies against Eimeria tenella sporozoites. Vet Immunol Immunopathol 121(1–2):58–67. https://doi.org/10.1016/j.vetimm.2007.08.005
Adams GP, Schier R (1999) Generating improved single-chain Fv molecules for tumor targeting. J Immunol Methods 231(1–2):249–260. https://doi.org/10.1016/S0022-1759(99)00161-1
Alcocer MJC, Doyen C, Lee HA, Morgan MRA (2000) Properties of polyclonal, monoclonal, and recombinant antibodies recognizing the organophosphorus pesticide chlorpyrifos-ethyl. J Agric Food Chem 48(9):4053–4059. https://doi.org/10.1021/jf990917l
Arap MA (2005) Phage display technology—applications and innovations. Genet Mol Biol 28(1):1–9. https://doi.org/10.1590/S1415-47572005000100001
Aris A, Leblanc S (2011) Maternal and fetal exposure to pesticides associated to genetically modified foods in Eastern Townships of Quebec, Canada. Reprod Toxicol 31(4):528–533. https://doi.org/10.1016/j.reprotox.2011.02.004
Asensio L, González I, García T, Martín R (2008) Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control 19(1):1–8. https://doi.org/10.1016/j.foodcont.2007.02.010
Ballari RV, Martin A, Gowda LR (2013) Detection and identification of genetically modified EE-1 brinjal (Solenum melongena) by single, multiplex and SYBR® real-time PCR. J Sci Food Agric 93(2):340–347. https://doi.org/10.1002/jsfa.5764
Bravo A, Soberón M (2008) How to cope with insect resistance to Bt toxins? Trends Biotechnol 26(10):573–579. https://doi.org/10.1016/j.tibtech.2008.06.005
Bravo A, Gill SS, Soberón M (2007) Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49(4):423–435. https://doi.org/10.1016/j.toxicon.2006.11.022
Bravo A, Likitvivatanavong S, Gill SS, Soberón M (2011) Bacillus thuringiensis: a story of a successful bio-insecticide. Insect Biochem Mol Biol 41(7):423–431. https://doi.org/10.1016/j.ibmb.2011.02.006
Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64(5):625–635. https://doi.org/10.1007/s00253-004-1559-9
Dai H, Gao H, Zhao X, Dai L, Zhang X, Xiao N, Zhao R, Hemmingsen SM (2003) Construction and characterization of a novel recombinant single-chain variable fragment antibody against white spot syndrome virus from shrimp. J Immunol Methods 279(1–2):267–275. https://doi.org/10.1016/S0022-1759(03)00182-0
Deckers S, Braren I, Greunke K, Meyer N, Ruhl D, Bredehorst R, Spillner E (2009) Establishment of hapten-specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries. Biotechnol Appl Biochem 52(1):79–87. https://doi.org/10.1042/BA20080032
Dong S, Zhang CZ, Zhang X, Liu Y, Zhong J, Xie Y, Xu CX, Ding Y, Zhang LQ, Liu XJ (2016) Production and characterization of monoclonal antibody broadly recognizing Cry1 toxins using designed polypeptide as hapten. Anal Chem 88(14):7023–7032. https://doi.org/10.1021/acs.analchem.6b00429
Dong S, Zhang X, Liu Y, Zhang C, Xie Y, Zhong J, Xu C, Liu X (2017) Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model. Anal Bioanal Chem 409(8):1985–1994. https://doi.org/10.1007/s00216-016-0146-0
Garet E, Cabado AG, Vieites JM, González-Fernández A (2010) Rapid isolation of single-chain antibodies by phage display technology directed against one of the most potent marine toxins: palytoxin. Toxicon 55(8):1519–1526. https://doi.org/10.1016/j.toxicon.2010.03.005
Giovannoli C, Anfossi L, Baggiani C, Giraudi G (2008) Binding properties of a monoclonal antibody against the Cry1Ab from Bacillus thuringiensis for the development of a capillary electrophoresis competitive immunoassay. Anal Bioanal Chem 392(3):385–393. https://doi.org/10.1007/s00216-007-1811-0
Golchin M, Aitken R (2008) Isolation by phage display of recombinant antibodies able to block adherence of Escherichia coli mediated by the K99 colonisation factor. Vet Immunol Immunopathol 121(3–4):321–331. https://doi.org/10.1016/j.vetimm.2007.10.005
Gram H, Marconi L-A, Barbas CF III, Collet TA, Lerner RA, Kang A (1992) In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library. Proc Natl Acad Sci U S A 89(8):3576–3580. https://doi.org/10.1073/pnas.89.8.3576
Hust M, Steinwand M, Al-Halabi L, Helmsing S, Schirrmann T, Dübel S (2009) Improved microtitre plate production of single chain Fv fragments in Escherichia coli. New Biotechnol 25(6):424–428. https://doi.org/10.1016/j.nbt.2009.03.004
Irving RA, Kortt AA, Hudson PJ (1996) Affinity maturation of recombinant antibodies using E. coli mutator cells. Immunotechnology 2(2):127–143. https://doi.org/10.1016/1380-2933(96)00044-9
Kamle S, Ali S (2013) Genetically modified crops: detection strategies and biosafety issues. Gene 522(2):123–132. https://doi.org/10.1016/j.gene.2013.03.107
Kamle S, Kumar A, Bhatnagar R (2011) Development of multiplex and construct specific PCR assay for detection of Cry2Ab transgene in genetically modified crops and product. GM Crops 2(1):74–81. https://doi.org/10.4161/gmcr.2.1.16017
Korpimäki T, Rosenberg J, Virtanen P, Karskela T, Lamminmäki U, Tuomola M, Vehniäinen M, Saviranta P (2002) Improving broad specificity hapten recognition with protein engineering. J Agric Food Chem 50(15):4194–4201. https://doi.org/10.1021/jf0200624
Korpimäki T, Rosenberg J, Virtanen P, Lamminmäki U, Tuomola M, Saviranta P (2003) Further improvement of broad specificity hapten recognition with protein engineering. Protein Eng 16(1):37–46. https://doi.org/10.1002/dvdy.21757
Korpimäki T, Brockmann EC, Kuronen O, Saraste M, Lamminmäki U, Tuomola M (2004) Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level. J Agric Food Chem 52(1):40–47. https://doi.org/10.1021/jf034951i
Kumar R (2011) A real-time immuno-PCR assay for the detection of transgenic Cry1Ab protein. Eur Food Res Technol 234(1):101–108. https://doi.org/10.1007/s00217-011-1618-2
Leivo J, Chappuis C, Lamminmäki U, Lövgren T, Vehniäinen M (2011) Engineering of a broad-specificity antibody: detection of eight fluoroquinolone antibiotics simultaneously. Anal Biochem 409(1):14–21. https://doi.org/10.1016/j.ab.2010.09.041
Li X, Li P, Lei J, Zhang Q, Zhang W, Li C (2013) A simple strategy to obtain ultra-sensitive single-chain fragment variable antibodies for aflatoxin detection. RSC Adv 3(44):22367–22372. https://doi.org/10.1039/c3ra42706d
Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths A, Winter G (1991) By-passing immunization: human antibodies from V-gene libraries displayed on phage. J Mol Biol 222(3):581–597. https://doi.org/10.1016/0022-2836(91)90498-U
Miler KD, Weaver-Feldhaus J, Gray SA, Siegel RW, Feldhaus MJ (2005) Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiac, Pichia pastoris, and Escherichia coli. Protein Expr Purif 42(2):255–267. https://doi.org/10.1016/j.pep.2005.04.015
Moreno M, Plana E, Manclús J, Montoya A (2014) Comparative study of monoclonal and recombinant antibody-based immunoassays for fungicide analysis in fruit juices. Food Anal Methods 7(2):481–489. https://doi.org/10.1007/s12161-013-9655-z
Pande J, Szewczyk MM, Grover AK (2010) Phage display: concept, innovations, applications and future. Biotechnol Adv 28(6):849–858. https://doi.org/10.1016/j.biotechadv.2010.07.004
Pardo-López L, Muñoz-Garay C, Porta H, Rodríguez-Almazán C, Soberón M, Bravo A (2009) Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis. Peptides 30(3):589–595. https://doi.org/10.1016/j.peptides.2008.07.027
Paul V, Steinke K, Meyer HHD (2008) Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810). Anal Chim Acta 607(1):106–113. https://doi.org/10.1016/j.aca.2007.11.022
Pigott CR, King MS, Ellar DJ (2008) Investigating the properties of Bacillus thuringiensis Cry proteins with novel loop replacements created using combinatorial molecular biology. Appl Environ Microbiol 74(11):3497–3511. https://doi.org/10.1128/AEM.02844-07
Ramirez-Romero R, Desneux N, Chaufaux J, Kaiser L (2008) Bt-maize effects on biological parameters of the non-target aphid Sitobion avenae (Homoptera: Aphididae) and Cry1Ab toxin detection. Pesticide Biochem Phys 91(2):110–115. https://doi.org/10.1016/j.pestbp.2008.01.010
Randhawa GJ, Singh M, Chhabra R, Sharma R (2010) Qualitative and quantitative molecular testing methodologies and traceability systems for commercialized Bt cotton events and other Bt crops under field trials in India. Food Anal Methods 3(4):295–303. https://doi.org/10.1007/s12161-010-9126-8
Roda A, Mirasoli M, Guardigli M, Michelini E, Simoni P, Magliulo M (2006) Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize. Anal Bioanal Chem 384(6):1269–1275. https://doi.org/10.1007/s00216-006-0308-6
Schlick JL, Desvoyes B, Hakil M, Adami P, Dulieu P (1999) Development of a double sandwich ELISA able to discriminate between free PAP (pokeweed antiviral protein) and complexed PAP in leaf extracts. Plant Sci 140(1):1–8. https://doi.org/10.1016/S0168-9452(98)00208-8
Shan GM, Embrey SK, Schafer B (2007) A highly specific enzyme-linked immunosorbent assay for the detection of Cry1Ac insecticidal crystal protein in transgenic WideStrike cotton. J Agric Food Chem 55(15):5974–5979. https://doi.org/10.1021/jf070664t
Shi XZ, Karkut T, Chamankhah M, Alting-Mees M, Hemmingsen SM, Hegedus D (2003) Optimal conditions for the expression of a singlechain antibody (scFv) gene in Pichia pastoris. Protein Expr Purif 28(2):321–330. https://doi.org/10.1016/S1046-5928(02)00706-4
Strachan G, McElhiney J, Drever MR, McIntosh F, Lawton LA, Porter AJ (2002) Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in Escherichia coli. FEMS Microbiol Lett 210(2):257–261. https://doi.org/10.1111/j.1574-6968.2002.tb11190.x
Terzy V, Morcia C, Gorrini A, Stanca AM, Cegrí PR, Faccioli P (2005) DNA-based methods for identification and quantification of small grain cereal mixtures and fingerprinting of varieties. J Cereal Sci 41(3):213–220. https://doi.org/10.1016/j.jcs.2004.08.003
Vachon V, Laprade R, Schwartz JL (2012) Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: a critical review. J Invertebr Pathol 111(1):1–12. https://doi.org/10.1016/j.jip.2012.05.001
Vázquez-Padrón RI, Gonzáles-Cabrera J, García-Tovar C, Neri-Bazan L, Lopéz-Revilla R, Hernández M, Moreno-Fierro L, de la Riva GA (2000) Cry1Ac protoxin from Bacillus thuringiensis sp., kurstaki HD-73 binds to surface proteins in the mouse small intestine. Biochem Biophys Res Commun 271(1):54–58. https://doi.org/10.1006/bbrc.2000.2584
Wang Y, Zhang X, Zhang CZ, Liu Y, Liu XJ (2012) Isolation of single chain variable fragment (scFv) specific for Cry1C toxin from human single fold scFv libraries. Toxicon 60(8):1290–1297. https://doi.org/10.1016/j.toxicon.2012.08.014
Wark KL, Hudson PJ (2006) Latest technologies for the enhancement of antibody affinity. Adv Drug Deliv Rev 58(5–6):657–670. https://doi.org/10.1016/j.addr.2006.01.025
Weisser NE, Hall JC (2009) Applications of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnol Adv 27(4):502–520. https://doi.org/10.1016/j.biotechadv.2009.04.004
Woern A, Plueckthun A (2001) Stability engineering of antibody single-chain Fv fragments. J Mol Biol 305(5):989–1010. https://doi.org/10.1006/jmbi.2000.4265
Zhang M, Yang JX, Lin XM, Zhu CH, He JQ, Liu H, Lin TL (2010) A double antibody sandwich enzyme-linked immunosorbent assay for detection of soft-shelled turtle iridovirus antigens. J Virol Methods 167(2):193–198. https://doi.org/10.1016/j.jviromet.2010.04.004
Zhang X, Liu Y, Zhang CZ, Wang Y, Xu CX, Liu XJ (2012) Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt) Cry1B toxin. Ecotoxicol Environ Saf 81(1):84–90. https://doi.org/10.1016/j.ecoenv.2012.04.021
Zhang X, Xu C, Zhang C, Liu Y, Xie Y, Liu X (2014) Established a new double antibodies sandwich enzyme linked immunosorbent assay for detecting Bacillus thuringiensis (Bt) Cry1Ab toxin based single-chain variable fragments from a naïve mouse phage displayed library. Toxicon 81:13–22. https://doi.org/10.1016/j.toxicon.2014.01.010
Zhao Y, Amer S, Wang J, Wang C, Gao Y, Kang G, Bao Y, He H, Qin J (2010) Construction, screening and identification of a phage display antibody library against the Eimeria acervulina merozoite. Biochem Biophys Res Co 393(4):703–707. https://doi.org/10.1016/j.bbrc.2010.02.063
Zhu X, Chen L, Shen P, Jia J, Zhang D, Yang L (2011) High sensitive detection of Cry1Ab protein using a quantum dot-based fluorescence-linked immunosorbant assay. J Agric Food Chem 59(6):2184–2189. https://doi.org/10.1021/jf104140t
Zou L, Xu Y, Li Y, He Q, Chen B, Wang D (2014) Development of a single-chain variable fragment antibody-based enzyme-linked immunosorbent assay for determination of fumonisin B1 in corn samples. J Sci Food Agric 94(9):1865–1871. https://doi.org/10.1002/jsfa.6505
Acknowledgements
This research was supported by the General Financial Grant from the China Postdoctoral Science Foundation (No. 2017M621674), the Special Fund for Agro-scientific Research in the Public Interest (201303088), the Natural Science Foundation of Jiangsu Province (BK20131333), and the Basic Research Project (Natural Science Foundation for Young Scholars) of Jiangsu Province, China (Grant No. BK20170489).
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Dong, S., Bo, Z., Zhang, C. et al. Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library. Appl Microbiol Biotechnol 102, 3363–3374 (2018). https://doi.org/10.1007/s00253-018-8797-8
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DOI: https://doi.org/10.1007/s00253-018-8797-8