In vitro evolution of myc-tag antibodies: in-depth specificity and affinity analysis of Myc1-9E10 and Hyper-Myc
- Russo, G., Unkauf, T., Meier, D., Wenzel, E.V., Langreder, N., Schneider, K.T., Wiesner, R., Bischoff, R., Stadler, V., Dübel, S.
- Biological chemistry 403(5-6): 479-494 (Journal)
- Registered Authors
- Russo, Giulio
- epitope mapping, hypermyc, myc-tag, peptide microarrays, phage display, recombinant antibody
- MeSH Terms
- Antibodies, Monoclonal*/chemistry
- Epitope Mapping
- Proto-Oncogene Proteins c-myc/genetics
- 35312243 Full text @ Biol. Chem.
Russo, G., Unkauf, T., Meier, D., Wenzel, E.V., Langreder, N., Schneider, K.T., Wiesner, R., Bischoff, R., Stadler, V., Dübel, S. (2022) In vitro evolution of myc-tag antibodies: in-depth specificity and affinity analysis of Myc1-9E10 and Hyper-Myc. Biological chemistry. 403(5-6):479-494.
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
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Engineered Foreign Genes
Errata and Notes