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Cited by 44Monoclonal and Polyclonal Antibodies ResearchProtein purification and stabilityRNA and protein synthesis mechanisms

Ecobody technology: Rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation

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Ojima-Kato, Teruyo, Nagai, Satomi, Nakano, Hideo·2017·Scientific Reports
DOI 10.1038/s41598-017-14277-0PMC PMC5656612
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Abstract

We report a rapid and cost-effective monoclonal antibody screening method from single animal B cells using reverse transcription (RT)-PCR and Escherichia coli cell-free protein synthesis (CFPS), which allows evaluation of antibodies within 2 working days. This process is named “Ecobody technology”. The method includes strategies to isolate B cells that specifically bind an antigen from the peripheral blood of immunised animals, and single-cell RT-PCR to generate DNA fragments of the V H and V L genes, followed by CFPS for production of fragments of antigen binding (Fab). In the CFPS step, we employed our techniques: 1) ‘Zipbody’ as a method for producing Fab, in which the association of heavy and light chains is facilitated by adhesive leucine zipper peptides fused at the C-termini of the Fab; and 2) an N-terminal SKIK peptide tag that can increase protein expression levels. Using Ecobody technology, we obtained highly-specific monoclonal antibodies for the antigens Vibrio parahaemolyticus and E. coli O26. The anti-V. parahaemolyticus Zipbody mAb was further produced in E. coli strain SHuffle T7 Express in inclusion bodies and refolded by a conventional method, resulting in significant antigen-binding activity (KD = 469 pM) and productivity of 8.5 mg purified antibody/L-culture.

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  • The Ecobody technology enables efficient and cost-effective production of functional proteins including monoclonal antibodies and related fragments.

    “The well-known and innovative Ecobody technology [179] enables an efficient and cost-effective production of functional proteins including monoclonal antibodies and related fragments [76,180,181,182].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [179]

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  1. [1]Köhler G Milstein C Continuous cultures of fused cells secreting antibody of predefined specificity Nature 1975 256 495 497 10.1038/256495a0 1172191 · DOI
  2. [2]Yu X McGraw PA House FS Crowe JE An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies J. Immunol. Methods 2008 336 142 151 10.1016/j.jim.2008.04.008 18514220 PMC2519117 · DOI
  3. [3]Bradbury AR Sidhu S Dübel S McCafferty J Beyond natural antibodies: the power of in vitro display technologies Nat. Biotechnol. 2011 29 245 254 10.1038/nbt.1791 21390033 PMC3057417 · DOI
  4. [4]Smith K Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen Nat. Protoc. 2009 4 372 384 10.1038/nprot.2009.3 19247287 PMC2750034 · DOI
  5. [5]Tiller T Single B cell antibody technologies New Biotechnol. 2011 28 453 457 10.1016/j.nbt.2011.03.014 PMC7102800 21473940 · DOI
  6. [6]Kurosawa N Yoshioka M Fujimoto R Yamagishi F Isobe M Rapid production of antigen-specific monoclonal antibodies from a variety of animals BMC Biol. 2012 10 80 10.1186/1741-7007-10-80 23017270 PMC3520816 · DOI
  7. [7]Seeber S A robust high throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood PLoS ONE 2014 9 e86184 10.1371/journal.pone.0086184 24503933 PMC3913575 · DOI
  8. [8]Starkie DO Compson JE Rapecki S Lightwood DJ Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells PLoS ONE 2016 11 e0152282 10.1371/journal.pone.0152282 27022949 PMC4811437 · DOI
  9. [9]Cox KS Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures MAbs 2016 8 129 140 10.1080/19420862.2015.1109757 26491897 PMC4966506 · DOI
  10. [10]Ohashi H Kanamori T Shimizu Y Ueda T A highly controllable reconstituted cell-free system–a breakthrough in protein synthesis research Curr. Pharm. Biotechnol. 2010 11 267 271 10.2174/138920110791111889 20210745 · DOI
  11. [11]Carlson ED Gan R Hodgman CE Jewett MC Cell-free protein synthesis: applications come of age Biotechnol. Adv. 2012 30 1185 1194 10.1016/j.biotechadv.2011.09.016 22008973 PMC4038126 · DOI
  12. [12]Kanter G Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines Blood 2007 109 3393 3399 10.1182/blood-2006-07-030593 17164345 PMC1852255 · DOI
  13. [13]Zawada JF Microscale to manufacturing scale-up of cell-free cytokine production–a new approach for shortening protein production development timelines Biotechnol. Bioeng. 2011 108 1570 1578 10.1002/bit.23103 21337337 PMC3128707 · DOI
  14. [14]Samuel PP Smith LP Phillips GN Olson JS Apoglobin stability is the major factor governing both cell-free and in vivo expression of holomyoglobin J. Biol. Chem. 2015 290 23479 95 10.1074/jbc.M115.672204 26205820 PMC4583012 · DOI
  15. [15]Jiang X A novel strategy for generation of monoclonal antibodies from single B cells using rt-PCR technique and in vitro expression Biotechnol. Prog. 2006 22 979 988 10.1021/bp060092h 16889373 · DOI · Paper page
  16. [16]Sabrina Y Ali M Nakano H In vitro generation of anti-hepatitis B monoclonal antibodies from a single plasma cell using single-cell RT-PCR and cell-free protein synthesis J. Biosci. Bioeng. 2010 109 75 82 10.1016/j.jbiosc.2009.07.001 20129087 · DOI · Paper page
  17. [17]Ojima-Kato T Hashimura D Kojima T Minabe S Nakano H In vitro generation of rabbit anti-Listeria monocytogenes monoclonal antibody using single cell based RT-PCR linked cell-free expression systems J. Immunol. Methods 2015 427 58 65 10.1016/j.jim.2015.10.001 26454028 · DOI · Paper page
  18. [18]Arai H Glabe C Luecke H Crystal structure of a conformation-dependent rabbit IgG Fab specific for amyloid prefibrillar oligomers Biochim. Biophys. Acta. 2012 1820 1908 1914 10.1016/j.bbagen.2012.08.016 22940003 PMC3973424 · DOI
  19. [19]Merk H Gless C Maertens B Gerrits M Cell-free synthesis of functional and endotoxin-free antibody Fab fragments by translocation into microsomes Biotechniques 2012 53 153 160 22963477
  20. [20]Stafford RL Matsumoto ML Yin G Cai Q In vitro Fab display: a cell-free system for IgG discovery Protein Eng. Des. Sel. 2014 27 97 109 10.1093/protein/gzu002 24586053 PMC3966677 · DOI
  21. [21]Groff D Engineering toward a bacterial ‘endoplasmic reticulum’ for the rapid expression of immunoglobulin proteins Mabs 2014 6 671 678 10.4161/mabs.28172 24517929 PMC4011911 · DOI
  22. [22]Cai Q A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell‐free protein synthesis systems Biotechnol. Prog. 2015 31 823 831 10.1002/btpr.2082 25826247 PMC5029582 · DOI
  23. [23]Ojima-Kato T ‘Zipbody’ leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems Protein Eng. Des. Sel. 2016 29 149 157 10.1093/protein/gzw001 26902097 · DOI · Paper page
  24. [24]Ojima-Kato T Nagai S Nakano H N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae J. Biosci. Bioeng. 2017 123 540 546 10.1016/j.jbiosc.2016.12.004 28052816 · DOI · Paper page
  25. [25]Concepcion J Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization Comb. Chem. High Throughput Screen. 2009 12 791 800 10.2174/138620709789104915 19758119 · DOI
  26. [26]Oh IS Lee JC Lee M Chung J Kim DM Cell-free production of functional antibody fragments Bioproc. Biosyst. Eng. 2010 33 127 132 10.1007/s00449-009-0372-3 19701777 · DOI
  27. [27]Clargo MA The rapid generation of recombinant functional monoclonal antibodies from individual, antigenspecific bone marrow-derived plasma cells isolated using a novel fluorescence-based method Mabs 2014 6 143 159 10.4161/mabs.27044 24423622 PMC3929438 · DOI
  28. [28]Tsumoto K Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent—application to a human single-chain Fv fragment J. Immunol. Methods 1998 219 119 129 10.1016/S0022-1759(98)00127-6 9831393 · DOI
  29. [29]Thomson C Olson M Jackson L Schrader J A Simplified Method for the Efficient Refolding and Purification of Recombinant Human GM-CSF Plos One 2012 7 e49891 10.1371/journal.pone.0049891 23166789 PMC3498172 · DOI
  30. [30]Fujii T Ohkuri T Onodera R Ueda T Stable Supply of Large Amounts of Human Fab from the Inclusion Bodies in E. coli J. Biochem. 2007 141 699 707 10.1093/jb/mvm075 17383978 · DOI
  31. [32]Landry JP Ke Y Yu GL Zhu XD Measuring affinity constants of 1450 monoclonal antibodies to peptide targets with a microarray-based label-free assay platform J. Immunol. Methods 2015 417 86 96 10.1016/j.jim.2014.12.011 25536073 PMC4339518 · DOI

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Authors
Ojima-Kato, T., Nagai, S., & Nakano, H.
Affiliations
Nagoya University (JP)
Corresponding author
Nakano, Hideo
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-10-25
Volume
7
Issue
1
Pages
13979-13979
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-017-14277-0
PMC
PMC5656612
OpenAlex
W2766510035
Cited by
44
References
1
Citations by year
2026: 9 · 2025: 9 · 2024: 8 · 2023: 5 · 2022: 6 · 2021: 1 · 2020: 3 · 2018: 3
FWCI
1.26
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Japan Society for the Promotion of Science (17J02835)
Topics
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
Keywords
monoclonal antibody screening, cell-free protein synthesis, antigen-binding fragments, single-cell RT-PCR, Vibrio parahaemolyticus, Escherichia coli O26, protein refolding
MeSH terms
Animals, Antibodies, Monoclonal, Antigens, Bacterial, B-Lymphocytes, Cell-Free System, Enzyme-Linked Immunosorbent Assay, Escherichia coli, Immunoglobulin Fab Fragments, Immunoglobulin Heavy Chains, Immunoglobulin Light Chains, Rabbits, Protein Biosynthesis, Vibrio parahaemolyticus, Protein Engineering

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