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Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing
Published on: September 25, 2011
Characterization of human colorectal cancer MDR1/P-gp Fab antibody
Xuemei Zhang1, Gary Guishan Xiao2, Ying Gao3
1The Medical College of Dalian University, Dalian Economic & Technical Development Zone, Dalian 116622, China ; Department of Biochemistry and Molecular Biology, Dalian Medical University, 9 Western Section, Lvshun South Street, Lvshunkou District, Dalian 116044, China.
Abstract:
In this study, the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, were isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21 and human colorectal cancer tissue homogenate, resulting in identification of an optimal recombinant Fab clone (Number 29). Further characterization by recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29 by Isopropyl β-D-1-thiogalactopyranoside (IPTG). After purified by HiTrap Protein L, the specificity of the Fab antibody to P-gp21 was also confirmed. Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope (ALKDKKELEGSGKIAT) comprising residues 883-898 within the transmembrane (TM) domain of human P-gp, but also the binding ability with it was verified. The clinical implication of our results for development of personalized therapy of colorectal cancer will be further studied.
Insights
Researchers developed a novel mouse monoclonal antibody Fab fragment targeting the P-gp21 protein to combat multidrug resistance. This antibody fragment shows potential for personalized colorectal cancer therapy by binding to a specific epitope on P-gp.
Area of Science:
- Immunology
- Molecular Biology
- Oncology
Background:
- Multidrug resistance protein P-gp (P-glycoprotein) is a key factor in the ineffectiveness of cancer chemotherapy.
- Developing targeted therapies against P-gp is crucial for improving treatment outcomes in cancers like colorectal cancer.
Purpose of the Study:
- To generate a novel mouse monoclonal antibody Fab fragment with biological activity against P-gp.
- To identify and characterize a specific epitope on P-gp for targeted therapeutic development.
Main Methods:
- Utilized phage display technology to create and select antibody Fab fragments against a 21 kDa peptide of P-gp.
- Employed enzyme-linked immunosorbent assay (ELISA) for screening and characterization of antibody binding and specificity.
- Recloned and induced Fab antibody expression using Isopropyl β-D-1-thiogalactopyranoside (IPTG) and purified using HiTrap Protein L.
Main Results:
- Identified an optimal recombinant Fab clone (Number 29) exhibiting binding and neutralized activity against P-gp21.
- Confirmed the specificity of the Fab antibody to P-gp21 and its recognition of human colorectal cancer tissue homogenate.
- Determined the targeted epitope as a 16-peptide sequence (ALKDKKELEGSGKIAT) within the transmembrane domain of human P-gp (residues 883-898).
Conclusions:
- Successfully generated a biologically active monoclonal antibody Fab fragment targeting a specific P-gp epitope.
- The findings suggest potential for developing personalized therapies for colorectal cancer by targeting P-gp.
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