Related Experiment Video
Updated: Sep 28, 2026
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
[Preparation of phage-displayed single chain variable fragments of monoclonal antibody MC5 recognizing colorectal
Feng-tian He1, Rong-fen Li, Yan Zhang
1Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing 400038, P. R. China. hefengtian@163.net
Background & Objective:
MC5 is a murine monoclonal antibody with a good specificity to human colorectal carcinoma and smaller murine antibody can significantly decrease the possibility of developing human antimouse antibody response in vivo study. The aim of this study was to prepare single chain variable fragments (ScFv) of MC5.
Methods:
mRNA was isolated from the hybridoma cell line producing MC5, and the DNAs encoding variable domains of heavy and light chains(VH and VL DNAs) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNAs with a linker DNA. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFvs. After two rounds of panning with cell line SW480 highly expressing MC5-binding antigen, the phage clones displayed ScFv fragments of the antibody were selected by ELISA, and the affinity of the positive phage clones was assayed by competition ELISA.
Results:
The VH, VL, and ScFv DNAs were about 340 bp, 320 bp, and 750 bp, respectively. Ten phage clones displayed ScFv of MC5 were selected from 25 enriched phage clones, and 3 of the 10 phage clones had higher affinity of binding to the antigen.
Conclusion:
The phage-displayed ScFv fragments of monoclonal antibody MC5 are successfully produced by phage display technique, which may provide a way for broadening the application range of the antibody.
Insights
Researchers successfully produced single chain variable fragments (ScFv) of the MC5 monoclonal antibody using phage display technology. This advancement in antibody engineering may expand the therapeutic applications of MC5 for colorectal carcinoma.
Area of Science:
- Immunotechnology
- Molecular Biology
- Biochemistry
Context:
- Monoclonal antibody MC5 exhibits high specificity for human colorectal carcinoma.
- Developing human antimouse antibody responses is a concern with murine antibodies.
- Smaller antibody formats can mitigate immunogenicity.
Purpose:
- To prepare single chain variable fragments (ScFv) of the MC5 monoclonal antibody.
- To engineer a smaller, potentially less immunogenic antibody derivative.
- To explore alternative antibody formats for colorectal carcinoma targeting.
Summary:
- Variable heavy (VH) and light (VL) chain domains of MC5 were amplified via RT-PCR.
- ScFv fragments were assembled, cloned into a phagemid vector, and expressed on phage.
- Phage display panning identified clones with high-affinity binding to the MC5 antigen.
Impact:
- Successfully produced phage-displayed ScFv fragments of MC5.
- Demonstrated the efficacy of phage display for antibody fragment engineering.
- Paved the way for broader applications of MC5 in colorectal cancer therapy.
