In vitro evolution of myc- tag antibodies: in-depth specificity and affinity analysis of Myc1-9E10 and Hyper-Myc

Biol Chem. 2022 Mar 22;403(5-6):479-494. doi: 10.1515/hsz-2021-0405. Print 2022 Apr 26.

Abstract

One of the most widely used epitope tags is the myc-tag, recognized by the anti-c-Myc hybridoma antibody Myc1-9E10. Combining error-prone PCR, DNA shuffling and phage display, we generated an anti-c-Myc antibody variant (Hyper-Myc) with monovalent affinity improved to 18 nM and thermal stability increased by 37%. Quantification of capillary immunoblots and by flow cytometry demonstrated improved antigen detection by Hyper-Myc. Further, three different species variants of this antibody were generated to allow the use of either anti-human, anti-mouse or anti-rabbit Fc secondary antibodies for detection. We characterized the specificity of both antibodies in depth: individual amino acid exchange mapping demonstrated that the recognized epitope was not changed by the in vitro evolution process. A laser printed array of 29,127 different epitopes representing all human linear B-cell epitopes of the Immune Epitope Database allowing to chart unwanted reactivities with mimotopes showed these to be very low for both antibodies and not increased for Hyper-Myc despite its improved affinity. The very low background reactivity of Hyper-Myc was confirmed by staining of myc-tag transgenic zebrafish whole mounts. Hyper-Myc retains the very high specificity of Myc1-9E10 while allowing myc-tag detection at lower concentrations and with either anti-mouse, anti-rabbit or anti human secondary antibodies.

Keywords: epitope mapping; hypermyc; myc-tag; peptide microarrays; phage display; recombinant antibody.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal* / chemistry
  • Epitope Mapping
  • Epitopes
  • Mice
  • Proto-Oncogene Proteins c-myc / genetics
  • Rabbits
  • Zebrafish*

Substances

  • Antibodies, Monoclonal
  • Epitopes
  • Proto-Oncogene Proteins c-myc