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2000
Volume 29, Issue 1
  • ISSN: 1386-2073
  • E-ISSN: 1875-5402

Abstract

Introduction/Objective

Antibodies have broad applications in various fields, such as biology and medicine. The screening and preparation of highly specific and sensitive antibodies are essential research areas. Several techniques for the preparation of mouse-derived antibodies have been developed, but limited studies on rabbit-derived antibodies with a broader antibody profile and easier humanization are reported. An improved yeast surface display technique was used for rapid screening of rabbit-derived Fab antibodies.

Methods

After RNA extraction from peripheral rabbit blood, a cDNA library was obtained by reverse transcription. After recombinant vector construction, the expressed sequence in the form of Fab antibody structure was fused to the N-terminal end of Aga2p in the vector; a bidirectional promoter was inserted and successfully expressed in brewer's yeast EBY100. In addition, sequences, such as leucine zipper and inulinase signal peptide (INU), were inserted into the recombinant vector to improve the expression and stability of Fab antibody further.

Results

A biotin-labeled salbutamol marker was synthesized, and two rabbit-derived salbutamol-Fab antibodies were screened in three weeks using fluorescence-activated cell sorting (FACS).

Conclusion

After antigen-binding kinetic studies, the screened antibodies demonstrated good affinity and specificity.

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2026-02-19
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References

  1. SujanK. DharM.D. Engineering antibodies.J. Indian Inst. Sci.2018981
    [Google Scholar]
  2. ManabeS. YamaguchiY. Antibody glycoengineering and homogeneous antibody‐drug conjugate preparation.Chem. Rec.202121113005301410.1002/tcr.202100054 33886147
    [Google Scholar]
  3. MadanB. ReddemE.R. WangP. CasnerR.G. NairM.S. HuangY. FahadA.S. de SouzaM.O. BanachB.B. López AcevedoS.N. PanX. NimraniaR. TengI.T. BahnaF. ZhouT. ZhangB. YinM.T. HoD.D. KwongP.D. ShapiroL. DeKoskyB.J. Antibody screening at reduced pH enables preferential selection of potently neutralizing antibodies targeting SARS‐COV ‐2.AIChE J.20216712e1744010.1002/aic.17440 34898670
    [Google Scholar]
  4. ParolaC. NeumeierD. ReddyS.T. Integrating high‐throughput screening and sequencing for monoclonal antibody discovery and engineering.Immunology20181531314110.1111/imm.12838 28898398
    [Google Scholar]
  5. IlinykhP.A. GraberJ. KuzminaN.A. HuangK. KsiazekT.G. CroweJ.E.Jr BukreyevA. Ebolavirus chimerization for the development of a mouse model for screening of bundibugyo-specific antibodies.J. Infect. Dis.2018218Suppl. 5S418S422 30060231
    [Google Scholar]
  6. KatoM. HanyuY. Single-step colony assay for screening antibody libraries.J. Biotechnol.20172551810.1016/j.jbiotec.2017.06.010 28641985
    [Google Scholar]
  7. BieZ. XingR. HeX. MaY. ChenY. LiuZ. Precision imprinting of glycopeptides for facile preparation of glycan-specific artificial antibodies.Anal. Chem.201890169845985210.1021/acs.analchem.8b01903 30036038
    [Google Scholar]
  8. YanevaM.Y. IvanovY.L. GodjevargovaT.I. Preparation of polyclonal antibodies with application for an organophosphorus pesticide immunoassay.Anal. Lett.20175081307132410.1080/00032719.2016.1221417
    [Google Scholar]
  9. LaustsenA.H. GreiffV. Karatt-VellattA. MuyldermansS. JenkinsT.P. Animal immunization, in vitro display technologies, and machine learning for antibody discovery.Trends Biotechnol.202139121263127310.1016/j.tibtech.2021.03.003 33775449
    [Google Scholar]
  10. PrabakaranP. RaoS.P. WendtM. Animal immunization merges with innovative technologies: A new paradigm shift in antibody discovery.MAbs2021131192434710.1080/19420862.2021.1924347 33947305
    [Google Scholar]
  11. GreenfieldE.A. Standard immunization of rabbits. Cold Spring Harb. Protoc.,202020209pdb.prot10030510.1101/pdb.prot100305 32873729
    [Google Scholar]
  12. RossiS. LaurinoL. FurlanettoA. ChinellatoS. OrvietoE. CanalF. FacchettiF. Dei TosA.P. Rabbit monoclonal antibodies: A comparative study between a novel category of immunoreagents and the corresponding mouse monoclonal antibodies.Am. J. Clin. Pathol.2005124229530210.1309/NR8HN08GDPVEMU08 16040303
    [Google Scholar]
  13. Spieker-PoletH. SethupathiP. YamP.C. KnightK.L. Rabbit monoclonal antibodies: Generating a fusion partner to produce rabbit-rabbit hybridomas.Proc. Natl. Acad. Sci. USA199592209348935210.1073/pnas.92.20.9348 7568130
    [Google Scholar]
  14. GaryC. Making and Using Antibodies: A Practical Handbook2nd ed; CRC Press,2013729
    [Google Scholar]
  15. WeberJ. PengH. RaderC. From rabbit antibody repertoires to rabbit monoclonal antibodies.Exp. Mol. Med.2017493e30510.1038/emm.2017.23 28336958
    [Google Scholar]
  16. KennedyP.J. OliveiraC. GranjaP.L. SarmentoB. Monoclonal antibodies: Technologies for early discovery and engineering.Crit. Rev. Biotechnol.201838339440810.1080/07388551.2017.1357002 28789584
    [Google Scholar]
  17. PhuaS.X. ChanK.F. SuC.T.T. PohJ.J. GanS.K.E. Perspective: The promises of a holistic view of proteins—impact on antibody engineering and drug discovery.Biosci. Rep.2019391BSR2018195810.1042/BSR20181958 30630879
    [Google Scholar]
  18. SargunasP.R. SpanglerJ.B. Full speed AHEAD in antibody discovery.Nat. Chem. Biol.202117101011101210.1038/s41589‑021‑00838‑y 34211163
    [Google Scholar]
  19. GaaR. Menang-NdiE. PratapaS. NguyenC. KumarS. DoernerA. Versatile and rapid microfluidics-assisted antibody discovery.MAbs2021131197813010.1080/19420862.2021.1978130 34586015
    [Google Scholar]
  20. BoderE.T. WittrupK.D. Yeast surface display for screening combinatorial polypeptide libraries.Nat. Biotechnol.199715655355710.1038/nbt0697‑553 9181578
    [Google Scholar]
  21. SalemaV. FernándezL.Á. Escherichia coli surface display for the selection of nanobodies.Microb. Biotechnol.20171061468148410.1111/1751‑7915.12819 28772027
    [Google Scholar]
  22. ElisonG.L. XueY. SongR. AcarM. Insights into bidirectional gene expression control using the canonical GAL1/GAL10 promoter.Cell Rep.2018253737748.e410.1016/j.celrep.2018.09.050 30332652
    [Google Scholar]
  23. PartowS. SiewersV. BjørnS. NielsenJ. MauryJ. Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae.Yeast2010271195596410.1002/yea.1806 20625983
    [Google Scholar]
  24. UritT. LöserC. WunderlichM. BleyT. Formation of ethyl acetate by Kluyveromyces marxianus on whey: Studies of the ester stripping.Bioprocess Biosyst. Eng.201134554755910.1007/s00449‑010‑0504‑9 21191616
    [Google Scholar]
  25. PalY. KhushooA. MukherjeeK.J. Process optimization of constitutive human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expression in Pichia pastoris fed-batch culture.Appl. Microbiol. Biotechnol.200669665065710.1007/s00253‑005‑0018‑6 15983807
    [Google Scholar]
  26. ChungB.H. NamS.W. KimB.M. ParkY.H. Highly efficient secretion of heterologous proteins from Saccharomyces cerevisiae using inulinase signal peptides.Biotechnol. Bioeng.199649447347910.1002/(SICI)1097‑0290(19960220)49:4<473:AID‑BIT15>3.0.CO;2‑B 18623603
    [Google Scholar]
  27. SivelleC. SierockiR. Ferreira-PintoK. SimonS. MaillereB. NozachH. Fab is the most efficient format to express functional antibodies by yeast surface display.MAbs201810572072910.1080/19420862.2018.1468952 29708852
    [Google Scholar]
  28. GuptaS.K. ShuklaP. Microbial platform technology for recombinant antibody fragment production: A review.Crit. Rev. Microbiol.2017431314210.3109/1040841X.2016.1150959 27387055
    [Google Scholar]
  29. AlberT. Structure of the leucine zipper.Curr. Opin. Genet. Dev.19922220521010.1016/S0959‑437X(05)80275‑8 1638114
    [Google Scholar]
  30. BarnesB.E. JenkinsT.A. SteinL.M. MathersR.T. WicaksanaM. PasquinelliM.A. SavinD.A. Synthesis and characterization of a leucine-based block co-polypeptide: The effect of the leucine zipper on self-assembly.Biomacromolecules20202162463247210.1021/acs.biomac.0c00420 32378896
    [Google Scholar]
  31. BogenJ.P. StorkaJ. YanakievaD. FiebigD. GrzeschikJ. HockB. KolmarH. Isolation of common light chain antibodies from immunized chickens using yeast biopanning and fluorescence‐activated cell sorting.Biotechnol. J.2021163200024010.1002/biot.202000240 32914549
    [Google Scholar]
  32. WagnerJ.M. LiuL. YuanS.F. VenkataramanM.V. AbateA.R. AlperH.S. A comparative analysis of single cell and droplet-based FACS for improving production phenotypes: Riboflavin overproduction in Yarrowia lipolytica.Metab. Eng.20184734635610.1016/j.ymben.2018.04.015 29698778
    [Google Scholar]
  33. DevarajN.K. FinnM.G. Introduction: Click chemistry.Chem. Rev.2021121126697669810.1021/acs.chemrev.1c00469 34157843
    [Google Scholar]
  34. HamamichiS. FukuharaT. HattoriN. Immunotoxin screening system: A rapid and direct approach to obtain functional antibodies with internalization capacities.Toxins2020121065810.3390/toxins12100658 33076544
    [Google Scholar]
  35. HanX. WangY. LiS. HuC. LiT. GuC. WangK. ShenM. WangJ. HuJ. WuR. MuS. GongF. ChenQ. GaoF. HuangJ. LongY. LuoF. SongS. LongS. HaoY. LiL. WuY. XuW. CaiX. GaoQ. ZhangG. HeC. DengK. DuL. NaiY. WangW. XieY. QuD. HuangA. TangN. JinA. A rapid and efficient screening system for neutralizing antibodies and its application for Sars-Cov-2.Front. Immunol.20211265318910.3389/fimmu.2021.653189 33828563
    [Google Scholar]
  36. JinZ. WangL. CaoD. ZouS. ChenC. KangH. SongQ. WangH. TangY. A new method for rapid screening of hybridoma cell clones secreting paired antibodies using sandwich cell surface fluorescence immunosorbent assay.Anal. Chim. Acta2021116333849310.1016/j.aca.2021.338493 34024420
    [Google Scholar]
  37. SunY. BanB. BradburyA. AnsariG.A.S. BlakeD.A. Combining yeast display and competitive facs to select rare hapten-specific clones from recombinant antibody libraries.Anal. Chem.201688189181918910.1021/acs.analchem.6b02334 27571429
    [Google Scholar]
  38. von BoehmerL. LiuC. AckermanS. GitlinA.D. WangQ. GazumyanA. NussenzweigM.C. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.Nat. Protoc.201611101908192310.1038/nprot.2016.102 27658009
    [Google Scholar]
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