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. 2017 May 31;18(6):1169.
doi: 10.3390/ijms18061169.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies From Recombinant Antibody Phage Display Libraries

Free PMC article

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies From Recombinant Antibody Phage Display Libraries

Antti Tullila et al. Int J Mol Sci. .
Free PMC article


Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library.

Keywords: fragment antigen-binding (Fab); hapten; high-throughput; mycotoxin; phage display; recombinant antibody.

Conflict of interest statement

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.


Figure 1
Figure 1
Amino acid sequences of variable regions of recombinant antibodies analysed with unweighted pair group method with arithmetic mean (UPGMA) using Geneious 6.1 software (Biomatters, Auckland, New Zealand). (A) Heavy chain variable regions of anti-MPA clones; (B) Light chain variable regions of anti-MPA clones; (C) Heavy chain variable regions of anti-OTA clones; (D) Light chain variable regions of anti-OTA clones. The elution strategy for each clone is indicated in parenthesis.
Figure 2
Figure 2
Chemical structures of the analytes used in the affinity measurements. Drawn with CambridgeSoft ChemBioDraw 12 (Perkin Elmer, Waltham, MA, USA). AcMPAG, mycophenolic acid acyl-glucuronide; MPAG, mycophenolic acid glucuronide; OTA, ochratoxin A and OTB, ochratoxin B.
Figure 3
Figure 3
Affinity and specificity determinations of the MPA- and OTA-binding antibody clones having the highest affinities measured with SPR. (A) anti-MPA antibody (MPA14) competition against MPA, AcMPAG, and MPAG; (B) Anti-OTA antibody (OTA47) competition against OTA and OTB. Data derived from duplicates. Drawn with OriginPro 2015 (OriginLab, Northampton, MA, USA).

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    1. Bradbury A., Plückthun A. Reproducibility: Standardize antibodies used in research. Nature. 2015;518:27–29. doi: 10.1038/518027a. - DOI - PubMed
    1. Boder E.T., Wittrup K.D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 1997;15:553–557. doi: 10.1038/nbt0697-553. - DOI - PubMed
    1. Hanes J., Pluckthun A. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA. 1997;94:4937–4942. doi: 10.1073/pnas.94.10.4937. - DOI - PMC - PubMed
    1. Ho M., Nagata S., Pastan I. Isolation of anti-CD22 Fv with high affinity by Fv display on human cells. Proc. Natl. Acad. Sci. USA. 2006;103:9637–9642. doi: 10.1073/pnas.0603653103. - DOI - PMC - PubMed
    1. McCafferty J., Griffiths A.D., Winter G., Chiswell D.J. Phage antibodies: Filamentous phage displaying antibody variable domains. Nature. 1990;348:552–554. doi: 10.1038/348552a0. - DOI - PubMed

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