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Characterization of human colorectal cancer MDR1/P-gp Fab antibody.

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 ?-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.

SUBMITTER: Zhang X

PROVIDER: S-EPMC3856173 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Characterization of human colorectal cancer MDR1/P-gp Fab antibody.

Zhang Xuemei X Xiao Gary Guishan GG Gao Ying Y

TheScientificWorldJournal 20131107

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 ...[more]

PMID: 24348182

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Project description:ObjectiveTo analyse the single-nucleotide polymorphisms (SNPs): ABCB1(1236C>T), ABCB1(2677G>T/A), ABCB1(3435C>T) and haplotypes in the ABCB1/MDR1 gene, which could contribute to genetic risk of colorectal cancer (CRC). Disease association between the ABCB1/MDR1 genotype, allele, haplotype frequencies and histological features, such as TNM classification, localization of primary carcinoma, grade of malignancy, histological type of tumour, lymphoid infiltration and vessel invasion were estimated. In this study, the potential role of SNPs of the ABCB1/MDR1 gene as a prognostic marker for CRC was analysed.Materials and methodsTumour specimens of 95 patients with CRC were studied. Using automated sequencing or PCR-RFLP method, DNA for three common SNPs of ABCB1/MDR1 was extracted and analysed. The results of genotyping and haplotype analysis with histopathological features, grading and clinical staging of neoplasms were correlated.ResultsA statistically significant higher frequency of T(1236) allele in T1/T2 (89.7%), M0 groups (81.6%) and I/II clinical staging (82.7%) in comparison with T3/T4 (68.2%), M1 groups (47.4%) and III/IV clinical staging (65.1%) was detected. Furthermore, multivariate analysis according to Cox's proportional hazard model indicated that the T(1236) allele is a good, independent prognostic factor and the presence of this allele decreases the risk of death in comparison with a group without this allele (HR = 0.26; p = 0.0424). In addition, a statistically significant higher frequency of C(3435) allele and significant differences in the C(3435) allele distribution in N1/N2 group (91.7% and 62.5%, respectively) than N0 group (71.2% and 44.9%, respectively) was found. Each of the eight possible haplotypes was noted in M0 or I/II group and only seven in M1 or III/IV group. Haplotype T(1236)-G(2677)-C(3435) only in less advanced CRC subjects (9.6% in I/II and 9.2% in M0 group) was detected. In addition, significant differences in haplotype distributions between M0 or I/II and M1 or III/IV group were found (p = 0.01 and p = 0.05, respectively).ConclusionsThese results suggest association between T(1236) allele and T(1236)-G(2677)-C(3435) haplotype and less advanced CRC, so these genetic markers may play a role as potentially good prognostic factors. Differences in haplotype distributions and degree of clinical staging may suggest that some other potential SNPs, especially in regulatory region of ABCB1/MDR1 gene, may influence P-glycoprotein function and CRC progression.

Dataset Information

Characterization of human colorectal cancer MDR1/P-gp Fab antibody.

Publications

Characterization of human colorectal cancer MDR1/P-gp Fab antibody.

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