PUBLICATION

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

Authors
Russo, G., Unkauf, T., Meier, D., Wenzel, E.V., Langreder, N., Schneider, K.T., Wiesner, R., Bischoff, R., Stadler, V., Dübel, S.
ID
ZDB-PUB-220323-19
Date
2022
Source
Biological chemistry   403(5-6): 479-494 (Journal)
Registered Authors
Russo, Giulio
Keywords
epitope mapping, hypermyc, myc-tag, peptide microarrays, phage display, recombinant antibody
MeSH Terms
  • Animals
  • Antibodies, Monoclonal*/chemistry
  • Epitope Mapping
  • Epitopes
  • Mice
  • Proto-Oncogene Proteins c-myc/genetics
  • Rabbits
  • Zebrafish*
PubMed
35312243 Full text @ Biol. Chem.
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.
Genes / Markers
Figures
Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes