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

Scientific Reports
Teruyo Ojima-KatoHideo Nakano

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 VH and VL 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 (K D = 469 pM) and ...Continue Reading

References

Mar 27, 2007·Journal of Biochemistry·Testuro FujiiTadashi Ueda
Jun 3, 2008·Journal of Immunological Methods·Xiaocong YuJames E Crowe
Feb 28, 2009·Nature Protocols·Kenneth SmithPatrick C Wilson
Aug 25, 2009·Bioprocess and Biosystems Engineering·In-Seok OhDong-Myung Kim
Sep 18, 2009·Combinatorial Chemistry & High Throughput Screening·Joy ConcepcionHong Tan
Mar 10, 2010·Current Pharmaceutical Biotechnology·Hiroyuki OhashiTakuya Ueda
Mar 11, 2011·Nature Biotechnology·Andrew R M BradburyJohn McCafferty
Apr 9, 2011·New Biotechnology·Thomas Tiller
Oct 20, 2011·Biotechnology Advances·Erik D CarlsonMichael C Jewett
Sep 29, 2012·BMC Biology·Nobuyuki KurosawaMasaharu Isobe
Mar 4, 2014·Protein Engineering, Design & Selection : PEDS·Ryan L StaffordAaron K Sato
Jul 25, 2015·The Journal of Biological Chemistry·Premila P SamuelJohn S Olson
Aug 28, 2015·Nature Communications·Michael-Paul RobinsonMehmet Berkmen
Feb 24, 2016·Protein Engineering, Design & Selection : PEDS·Teruyo Ojima-KatoHideo Nakano

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Citations

Dec 7, 2018·Scandinavian Journal of Immunology·Ge LiGencheng Han
Sep 4, 2020·International Journal of Molecular Sciences·Annamaria SandomenicoMenotti Ruvo

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Methods Mentioned

BETA
mammalian cell display
transfection
enzyme-linked immunosorbent assay
ELISA
density gradient centrifugation
fluorescence-activated cell sorting
PCR
electrophoresis
FACS
biolayer interferometry

Software Mentioned

Ecobody
image J
Zipbody

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