• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.829-832
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• 2001

Phage antibody 생산방법은 유전자 조작에 의하여 phage coat에 항체를 발현시키는 방법이며 그 library를 이용하여 일반 hybridoma 방법에 의해 항체를 생산하기 어려운 성분 (예: 독성물질, 면역화가 잘 안되는 물질 등) 에 대해 적용할 수 있는 장점을 제공한다. 본 연구진은 Griffin.1 antibody library의 positive control을 통해 phage 표면에 항체가 발현되는 지의 여부를 ELISA test를 통하여 확인하였다. 또한 human serum albumin을 모델분석물질로 사용하여 $1{\sim}4$차까지의 bio-panning 을 실시하였고 각 단계에서 증폭된 phage를 가지고 ELISA test를 한 결과 신호가 점진적으로 증가하는 data를 얻을 수 있었다. 이것을 통해 phage library로부터 특정 항원에 대한 monoclonal antibody를 생산할 수 있다는 사실을 검증하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제13권1호
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• pp.377-381
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• 2012

The aim of this study was to screen for polypeptides binding specifically to LoVo human colorectal cancer cells using a phage-displayed peptide library as a targeting vector for colorectal cancer therapy. Human normal colorectal mucous epithelial cells were applied as absorber cells for subtraction biopanning with a c7c phage display peptide library. Positive phage clones were identified by enzyme-linked immunosorbent assay and immunofluorescence detection; amino acid sequences were deduced by DNA sequencing. After 3 rounds of screening, 5 of 20 phage clones screened positive, showing specific binding to LoVo cells and a conserved RPM motif. Specific peptides against colorectal cancer cells could be obtained from a phage display peptide library and may be used as potential vectors for targeting therapy for colorectal cancer.

• Journal of Microbiology
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• 제37권2호
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• pp.97-101
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• 1999

Random hexapeptide library on the surface of filamentous bacteriophage was constructed using the SurfZAP vector. The size of the library was approximately 105. The peptide insert was flanked by two cysteines to constrain the peptide structure with a disulfide bond. This library was screened for the topoisomerase II binding peptide. Dramatic enrichment of the fusion phage over the VCS M13 helper phage was demonstrated by bio-panning affinity selection.

• BMB Reports
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• 제48권9호
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• pp.489-494
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• 2015

The in vitro antibody discovery technologies revolutionized the generation of target-specific antibodies that traditionally relied on the humoral response of immunized animals. An antibody library, a large collection of diverse, pre-constructed antibodies, can be rapidly screened using in vitro display technologies such as phage display. One of the keys to successful in vitro antibody discovery is the quality of the library diversity. Antibody diversity can be obtained either from natural B-cell sources or by the synthetic methods that combinatorially generate random nucleotide sequences. While the functionality of a natural antibody library depends largely upon the library size, various other factors can affect the quality of a synthetic antibody library, making the design and construction of synthetic antibody libraries complicated and challenging. In this review, we present various library designs and diversification methods for synthetic antibody library. From simple degenerate oligonucleotide synthesis to trinucleotide synthesis to physicochemically optimized library design, the synthetic approach is evolving beyond the simple emulation of natural antibodies, into a highly sophisticated method that is capable of producing high quality antibodies suitable for therapeutic, diagnostic, and other demanding applications. [BMB Reports 2015; 48(9): 489-494]

• Journal of Microbiology and Biotechnology
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• 제21권2호
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• pp.197-203
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• 2011

Lactobacilli are normal constituents of the intestinal microbiota, and some strains show the capacity to bind to extracellular matrix proteins and components of the mucosal layer, which represents an adaptation to persist in this niche. A shotgun phage-display library of Lactobacillus casei BL23 was constructed and screened for peptides able to bind to fibronectin and collagen. Clones showing binding to these proteins were isolated, which encoded overlapping fragments of a putative transcriptional regulator (LCABL_29260), a hypothetical protein exclusively found in the L. casei/rhamnosus group (LCABL_01820), and a putative phage-related endolysin (LCABL_13470). The construction of different glutathione S-transferase (GST) fusions confirmed the binding activity and demonstrated that the three identified proteins could interact with fibronectin, fibrinogen, and collagen. The results illustrate the utility of phage display for the isolation of putative adhesins in lactobacilli. However, it remains to be determined whether the primary function of these proteins actually is adhesion to mucosal surfaces.

• Molecules and Cells
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• 제27권3호
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• pp.313-319
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• 2009

The dual-vector system-II (DVS-II), which allows efficient display of Fab antibodies on phage, has been reported previously, but its practical applicability in a phage-displayed antibody library has not been verified. To resolve this issue, we created two small combinatorial human Fab antibody libraries using the DVS-II, and isolation of target-specific antibodies was attempted. Biopanning of one antibody library, termed DVFAB-1L library, which has a $1.3{\times}10^7$ combinatorial antibody complexity, against fluorescein-BSA resulted in successful isolation of human Fab clones specific for the antigen despite the presence of only a single light chain in the library. By using the unique feature of the DVS-II, an antibody library of a larger size, named DVFAB-131L, which has a $1.5{\times}10^9$ combinatorial antibody complexity, was also generated in a rapid manner by combining $1.3{\times}10^7$ heavy chains and 131 light chains and more diverse anti-fluorescein-BSA Fab antibody clones were successfully obtained. Our results demonstrate that the DVS-II can be applied readily in creating phage-displayed antibody libraries with much less effort, and target-specific antibody clones can be isolated reliably via light chain promiscuity of antibody molecules.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1316-1319
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• 2006

An antibody displayed phage collection (SBAE-2R), screened from a human synthetic phage antibody library (Fab 21ox), was used for the determination of dextran. The dextran-binding affinity was determined by serologically specific transmission electron microscopy (TEM) and a paper dipstick assay. The phage collection was distributed over the dextrancoated grids with 39$\pm$25 phages/$\mu$m$^2$ on the grids. Phages were not seen on dextran-coated grids exposed to the Fab 2lox phage library. The phage collection (SBAE-2R) produced 54$\pm$3 color normalized intensity (N.I.) from 125 ppm to 1,000 ppm of dextran and 5$\pm$1 (N.I.) for 63 ppm of dextran in a paper dipstick assay. This research extends the analytical options for dextran analysis by antibody displayed phage with a minimum of equipment usage.

• IMMUNE NETWORK
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• 제2권2호
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• pp.91-95
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• 2002

Background: In order to characterize human antibodies with specificity for mite allergens at the molecular level, a scFv phage display library was constructed using peripheral blood mononuclear lymphocytes from an asthma patient allergic to mite as Ig gene sources. Methods: Immunoglobulin $V_H$ and V gene fragments were obtained by polymerase chain reaction, and randomly combined in pCANTAB-5E vector. The resulting human scFv phage display library had $3{\times}10^4$ independent clones, and biopanning was performed with house dust mite extracts. Results: Four scFv clones specific for house dust mite extract were isolated. Immunoblot assay showed that our clones reacted to 25 kDa and 50~60 kDa proteins with unknown identity in mite extracts. Sequence analysis indicated that two clones (b7 and c15) are identical, and all clones belong to human $V_H3$ subgroup. On the other hand, light chain usage was different in that two clones (a2 and b7 / c15) belonging to V ${\kappa}4$ subgroup, but a4 used V ${\kappa}1$ light chain gene. Conclusion: Our approach should facilitate provision of useful information on the antibody responses against allergens at the molecular level in humans.

• BMB Reports
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• 제44권4호
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• pp.244-249
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• 2011

The quality of a phage-displayed antibody library deteriorates with clonal variations, which are caused by differentially expressed Escherichia coli antibody genes. Using the human Fab SP114 against the pyruvate dehydrogenase complex-E2 (PDCE2), we created four E. coli TOP10F' clones with a pCMTG phagemid encoding Fab-pIII (pCMTG-Fab), Fd ($V_H+C_{H1}$)-pIII (pCMTG-Fd), or light chain (L) (pCMTG-L), or the vector only (pCMTG-${\Delta}Fab$) to investigate the effect of clonal variations in a defined manner. Compared to the others, the E. coli clone with pCMTG-Fab was growth retarded in liquid culture, but efficiently produced phage progenies by Ex12 helper phage superinfection. Our results suggest that an antibody library must be cultured for a short duration before helper phage superinfection, and that the Ex12 helper phage helped to alleviate the detrimental effect of clonal variation, at least in part, by preferentially increasing functional phage antibodies during phage amplification.

• Bulletin of the Korean Chemical Society
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• 제34권2호
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• pp.460-464
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• 2013

Single-chain variable fragments of antibodies (scFv) specific to 2,4-dinitrotoluene (DNT) were isolated from a phage library displaying synthetic human scFv fragments with 6 diversified complementary determining regions (CDRs). A DNT derivative that contained an extended amine group was synthesized and conjugated to the NHS-group that was linked to magnetic beads. Phages specific to the immobilized DNT derivatives were isolated from the library after 4 rounds of sequential binding and elution processes. The displayed scFv fragments from the isolated phages showed consensus CDR sequences. One DNT-specific scFv was expressed in E. coli and purified using Ni-affinity chromatography. The purified DNT-specific scFv binds specifically to the immobilized DNT-derivative with $K_D$ value of $6.0{\times}10^{-7}$ M. The scFv and DNT interaction was not disrupted by the addition of 4-nitrotoluene or benzoic acid. These data demonstrate that the screened scFv from the phage displayed library could be used for selective and sensitive detection of explosives such as TNT.