• Journal of Photoscience
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• v.12 no.3
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• pp.123-127
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• 2005

The polyclonal antibody was generated against the peptide fragment of 62 amino acid residues (D 181-T242) near the COOH-terminal region of the extracellular domain of epithelial-cell adhesion molecule (Ep-CAM) and shown to be able to recognize Ep-CAM in competitive ELISA. Then, sulforhodamine 101 acid chloride (so called Texas red), a fluorescence dye, was conjugated to the affinity-purified anti-Ep-CAM antibody utilizing the reaction between the aliphatic amines of antibody and the sulfonyl chloride of Texas red. The molar ratio of Texas red to antibody was estimated to be approximately 1.86 by measuring optical densities at 280 nm and 596 nm, implying that the two molecules of Texas red at most were conjugated to antibody. The anti-Ep-CAM antibody-Texas red conjugate was then used for immunohistochemistry of CT-26 murine colon carcinoma cells. Based upon the fluorescence microscope images, anti-Ep-CAM antibody is able to deliver Texas red specifically to the surface of CT-26 cells on which Ep-CAM was actively expressed. This result indicates that anti-Ep-CAM antibody could be useful for the tissue-specific delivery of photosensitizers via antigen-antibody interaction.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.77-86
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• 2001

Background: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell. Methods: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. Results: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patients captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. Conclusion: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.

• Molecules and Cells
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• v.39 no.6
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• pp.460-467
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• 2016

Bacteriophytochromes are phytochrome-like light-sensing photoreceptors that use biliverdin as a chromophore. To study the biochemical properties of the Deinococcus radiodurans bacteriophytochrome (DrBphP) protein, two anti-DrBphP mouse monoclonal antibodies (2B8 and 3H7) were generated. Their specific epitopes were identified in our previous report. We present here fine epitope mapping of these two antibodies by using truncation and substitution of original epitope sequences in order to identify minimized epitope peptides. The previously reported original epitope sequences for 2B8 and 3H7 were truncated from both sides. Our analysis showed that the minimal peptide sequence lengths for 2B8 and 3H7 antibodies were nine amino acids (RDPLPFFPP) and six amino acids (PGEIEE), respectively. We further characterized these peptides in order to investigate their reactivity after single deletion and single substitution of the original peptides. We found that single-substituted 2B8 epitope (RDPLPAFPP) and dual-substituted 3H7 epitope (PGEIAD) showed significantly increased reactivity. These two antibodies with high reactivity for the short modified peptide sequences are valueble for developing new peptide tags for protein research.

• Journal of Periodontal and Implant Science
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• v.38 no.4
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• pp.565-578
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• 2008

Purpose: Porphyromonas gingivalis(P. gingivalis) heat shock protein (HSP)60 may play a role in the immunopathogenesis of periodontitis as well as atherosclerosis by modulating autoimmune reaction due to its high level of sequence homology between bacteria and human counterpart. The purpose of this study was to identify immunodomiant epitope of P. gingivalis HSP60 that is reactive exclusively to the homologous bacteria without reacting with human HSP. Materials and methods: The present study was performed to identify the peptide specifically recognized by anti-P. gingivalis HSP60 monoclonal antibodies mono-reactive to P. gingivalis HSP60. Results: Four different hybridomas were cloned producing monoclonal IgG antibodies exclusively to P. gingivalis HSP60. Thirty seven synthetic peptides (20-mer with 5-amino acid overlapping) were synthesized. All of these peptide were subject to SDS-PAGE for immunblot analysis. One peptide (TVPGGGTTYIRAIAALEGLK) and the other peptide (TLVVNRLRGSLKICAVKAPG) were recognized by all and one of the four monoclonal antibodies, respectively, that reacted solely with P. gingivalis HSP60. Immunohistochemistry to identify the localization of the HSP60 in the diseased gingival tissues revealed that all of the four monoclonal antibodies were highly reacted with the diseased gingival tissue than normal gingival tissue. Conclusion: The P. gingivalis HSP60 peptides (TVPGGGTTYIRAIAALEGLK and TLVVNRLRGSLKICAVKAPG, respectively) are positively involved in the immunopathologic process of periodontal disease. The peptide may potentially be developed as vaccine candidates. Further investigations are under way to identify more clones producing monoclonal antibodies reactive to P. gingivalis HSP and to other periodontopathogenic bacteria as well, while maintaining specificities to human counterpart.

• Plant Disease and Agriculture
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• v.5 no.1
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• pp.27-33
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• 1999

Frost injury of crops is closely related to the epiphytic population dynamics of ice nucleation-active (INA) bacteria, and the injury can be reduced by decreasing the INA bacterial population. In order to predict the epiphytic population of INA bacteria on crops, a rapid and accurate detection method has to be developed. In the previous report, we produced some antibodies against INA proteins purified from the outer membrane of INA bacteria. However it was difficult to produce the antibodies because the purification procedures of the INA proteins were complicated, and the final yield was too low. We designed a specific peptide from the N-terminal region of INA protein by computer analysis and synthesized the peptide in vitro in this experiment. The peptide sequence was Asp-Ser-Por-Leu-Ser-Leu-His-Ala-Asp, that is corresponding to the highly conserved region in several INA proteins, with predicted beta turn, coiling, and hydrophilic region. A polyclonal anti-INA peptide antiserum produced specifically recognized INA bacteria as few as 10 colony-forming units (CFU) in the ELISA reactions and did not respond to other non-INA bacteria. Serological specificity of the anti-INA peptide antiserum will facilitate the forecasting of the INA bacterial population dynamics on crops.

• Biomedical Science Letters
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• v.7 no.3
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• pp.103-110
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• 2001

$\alpha$-Fetoprotein(AFP) is a good marker for the detection of several diseases such as hepatocellular carcinoma, gonadal germ cell tumor, gastric tumor, and Down's syndrome. In this study, we developed ELISA, using synthetic peptides corresponding to the epitopes of AFP. Five kinds of peptides were synthesized from AFP to produce antibodies in rats that recognize AFP in human plasma as well as amniotic fluid and do not cross-react with serum albumin. All five kinds of antibodies showed good reactivities with their peptide-keyhole limpet hemocyanin conjugates. Anti-synthetic peptide 1 (R-N-E-Y-G-I-A-S-I-L, 4-13) antibody, in particular, reacted well with AEP as well as synthetic peptide 1-KLH but not with human serum albumin. The binding affinity(Kd) was 2.7$\times$10$^{-9}$M for peptide 1 and 6.8$\times$10$^{-8}$M for AEP. The range for measurement of AFP was 10~1,000 ng/ml. The within-assay and between-assay coefficients of variance(CV) were 4.83% and 10.97%, respectively. In a sample of 31 sera and 33 amniotic fluids, there was a good correlation between AFP values determined in this assay and those in a commercial kit. These results indicate that the antibodies against synthetic peptides corresponding to the epitopes of AFP are highly specific to APP and synthetic peptide-based ELISA would be useful for the measurement of human AFP.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1998.11a
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• pp.150-151
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• 1998

Drug delivery to the brain is facilitated by the synthesis of chimeric peptides, where in neuropharmaceuticals are linked to a vector such as an antibody to the transferrin receptor that mediates transcytosis through the blood-brain barrier (BBB). When disulfide linkers are used in the chimeric peptide, it is crucial that the S-S bridge is stable during transit and that cleavage does not occur prematurely within endothelial cells, as the peptide drug moiety would then be sequestered by the BBB instead of passing through it. The present study addressed that problem. As a model drug a metabolically stable opioid peptide, [$^3$H]DALDA (Tyr-dArg-Phe-Lys-NH$_2$), was used. It was monobiotinylated with NHS-SS-biotin to yield bio-[$^3$H]DALDA. The biotinylated peptide was bound to the vector OX26-SA which is a covalent conjugate of OX26 and streptavidin (molar ratio = 1: 1). In vitro treatment of the chimeric peptide, bio-[$^3$H]DALDA/OX26-SA, with a reducing agent, dithiothreitol, released the labeled peptide from the vector by conversion of bio-[$^3$H]DALDA to the desbiotinylated derivative, desbio-[$^3$H]DALDA.

• Journal of Biosystems Engineering
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• v.37 no.6
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• pp.420-428
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• 2012

Purpose: Porcine proliferative enteropathy(PPE), caused by the obligate intracellular bacterium Lawsonia intracellularis, is a widely distributed disease throughout the world causing substantial economic loss. The bacterial pathogen invades the intestinal epithelial cells which causes hyperplasia of the infected cells and leads to the process of disease pathogenesis. For diagnosing PPE in a pig farm in earlier stage, a rapid diagnosing test equipment is needed for farmers. To test the equipment appropriately, we prepare the samples and antibodies for rapid detecting the Lawsonia intracellularis in pig feces. Methods : To prepare the PPE infected samples, we sampled PPE suspected pig feces in a pig farm. To manufacture a anti-Lawsonia intracellularis antibody for capturing the Lawsonia intracellularis, the rabbit-anti LsaA synthetic peptide polyclonal antibody was inoculated to rabbits. To select the couple of antibodies which is most well sandwiched with the bacteria, ELISA test was done with PPE infected ileum samples. Finally, to verify the PPE infected feces which would be used to test the rapid kit, PCR test was done on the sampled PPE suspected feces Results : The rabbit-anti LsaA synthetic peptide polyclonal antibody is developed, and is verified to capture the bacterial well through the fluorescence antibody test. Also, we found that the monoclonal antibody and the polyclonal antibody could be used as couples for sandwiching the bacteria. Finally, through the PCR test for samples of pig feces, we could prepare the 150 PPE positive samples and 50 PPE negative samples. Conclusions : The manufactured polyclonal antibody and the imported monoclonal antibody could be used to capture the bacteria using the sandwich techniques. Also, the prepared PPE infected negative and positive samples could be used to test the performance of the rapid kit to capture the bacterium Lawsonia intracellularis.

• BMB Reports
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• v.30 no.3
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• pp.177-181
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• 1997

We constructed a single-chain immunoglobulin in which the carboxyl end of the heavy chain variable domain is covalently joined to the amino terminus of the light chain variable domain via peptide linker and the carboxyl end of the light chain variable domain is linked to human ${\gamma}1$ Fc region through the hinge region. The molecule was expressed in Chinese hamster ovary cells, assembled into a dimeric molecule and secreted into the culture medium. The dimeric molecule (2E11) was purified from the culture supernatant by affinity chromatography on Protein G-Sepharose column. The size of the unreduced or reduced protein was the expected molecular weight of approximately 120 or 60 kDa, respectively, as assessed by SDS-polyacrylamide gel electrophoresis. The antigen-binding affinity of 2E11 was almost the same as that of a native antibody counterpart (CS131A), suggesting that the single-chain immunoglobulin may function like a native antibody.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.12
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• pp.4833-4837
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• 2015

Biomarkers for preclinical diagnosis of cancer are valuable tools for detection of malignant tumors at early stages in groups at risk and screening healthy people, as well as monitoring disease recurrence after treatment of cancer. However the complexity of the body's response to the pathological processes makes it virtually impossible to evaluate this response to the development of the disease using a single biomarker that is present in the serum at low concentrations. An alternative approach to standard biomarker analysis is called immunosignature. Instead of going after biomarkers themselves this approach rely on the analysis of the humoral immune response to molecular changes associated with the development of pathological processes. It is known that antibodies are produced in response to proteins expressed during cancer development. Accordingly, the changes in antibody repertoire associated with tumor growth can serve as biomarkers of cancer. Immunosignature is a highly sensitive method for antibody repertoire analysis utilizing high density peptide microarrays. In the present review we discuss modern methods for antibody detection, as well as describe the principles and applications of immunosignature in research and clinical practice.