• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.86-86
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• 1995

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently by the use of specific anti-receptor antibodies. A 14-mer peptide (from Phe102 to Leu115 of ${\beta}$2-adrenergic receptor) was synthesized and this peptide was coupled to carrier protein Keyhole Limpet Hemocyanin(KLH) by glutaraldehyde method. A 0.5mg of KLH-coupled peptide was emulsified with equal volume of complete Freund's adjuvant and injected via popliteal lymph node to each of the three Newzealnd White rabbits. Booster injections were repeated at 4 weeks interval for three times with incomplete Freund's adjuvants. One week after the final injection, serum was prepared from ear artery. Nonspecific immunoglobulins were removed by passing the serum through KLH-Sepharose 6B affinity matrix and further by incubation with bovine lung aceton powder. The titer of the antibody for synthetic peptide which was determined by enzyme linked immunosorbent assay(ELISA) was about l/l,000. The antibody produced in this study revealed 67kDa protein band in the western blot of partially purified guinea pig lung ${\beta}$-adrenergic receptor preparation. The antibody inhibited ${\beta}$-adrenergic antaginist [3H] Dihydroalprenolol binding to soluble ${\beta}$-adrenergic receptor by 25% while control sera did not show any inhibitory effects, The result of this study suggests that the peptide sequence selected in this study may play some important roles in adrenergic receptor-ligand interaction.

• BMB Reports
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• v.38 no.3
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• pp.290-293
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• 2005

In order to investigate the epitope of basic fibroblast growth factor (bFGF) and its immunogenicity, the epitopes of bFGF were screened from the phage display library with monoclonal antibody GF22, which can neutralize the bio-activity of bFGF. By three rounds of screening, the positive phage clones with bFGF epitopes were selected, which can effectively block the bFGF to bind with GF22. Sequence analysis showed that the epitopes shared a highly conservative sequence (Leu-Pro-Pro/Leu-Gly-His-Phe/Ile-Lys). The sequence of PPGHFK was located at 22-27 of the bFGF. The specific immuno-response of mouse could be highly induced by phage clones with the epitopes. And the anti-bFGF activity induced by LPGHFK was 3 times higher than the original sequence, which showed that the mimetic peptide LPLGHIK might be used as a tumor vaccine in the prevention and treatment of tumor.

• Parasites, Hosts and Diseases
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• v.60 no.1
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• pp.7-14
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• 2022

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

• YAKHAK HOEJI
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• v.50 no.1
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• pp.33-39
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• 2006

We have previously reported the minimum criteria that can be applied to evaluate efficacy and safety of a DNA vaccine with use of Streptococcus pneumoniae DNA vaccine (SPDNA). The SPDNA was formulated by inserting the DNA sequences that are codons specific for the carbohydrate epitope in the capsule of S. penumoniae by phage display peptide library. Administration of the SPDNA into mice induced both humoral and cell-mediated immunities. The induction was protective even in the absence of CD4+ T lymphocyte in mice. Profiles of cytokine and isotyping of antibody displayed tendency of the Th1. In continuation of these studies, we examined if the efficacy of the SPNDA was provoked by the peptide recognized by codons specific for the capsule. Results showed that the peptide vaccine formulae (SPP) induced protective antibody in mice as did the SPDNA. Involvement of the cell-mediated immunity was also determined. Possible side effects of autoimmune diseases such as myositis and C3a production and tumor-formation were undetectable in mice given 7 times of SPDNA vaccination during entire of 92 days. Even after the frequent immunization, immunogenicity of the SPDNA was observed as determined for antibody production, suggesting that there was no immunotolerance provoked. All together, these examining factors would be applied to measurement of a DNA vaccine safety regarding the immunopathological aspect.

• Journal of Photoscience
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• v.10 no.3
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• pp.237-240
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• 2003

The tetradecameric peptide (K47-K60) near the NH$_2$-terminal region of epithelial-cell adhesion molecule (Ep-CAM) was chosen as antigenic site and a polyclonal antibody was generated, which could recognize Ep-CAM from the mouse colon tissue or the colon cancer cell, CT-26, in Western blot analysis. Then, the fluorescein isothiocyanate (FITC), a fluorescence dye, was conjugated with the affinity purified Ep-CAM antibody using thiocyanate and the amino groups of FITC and antibody, respectively. The molar ratio of FITC to antibody was estimated approximately 1.86 to 1.00 by measuring the optical densities at 492 nm and 280 nm. Ep-CAM antibody-FITC conjugate was then used for immunohistochemistry of the CT-26 cells. Judging from the shapes formed by fluorescence, the Ep-CAM antibody could delivered FITC to the surface of cells in which Ep-CAM was expressed. This result implies that Ep-CAM antibody could be also used for the tissue-specific delivery of the photosensitizer to the target protein via antigen-antibody interaction.

• BMB Reports
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• v.38 no.5
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• pp.517-525
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• 2005

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.

• BMB Reports
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• v.52 no.6
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• pp.397-402
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• 2019

Middle East respiratory syndrome coronavirus (MERS-CoV) uses the spike (S) glycoprotein to recognize and enter target cells. In this study, we selected two epitope peptide sequences within the receptor binding domain (RBD) of the MERS-CoV S protein. We used a complex consisting of the epitope peptide of the MERS-CoV S protein and CpG-DNA encapsulated in liposome complex to immunize mice, and produced the monoclonal antibodies 506-2G10G5 and 492-1G10E4E2. The western blotting data showed that both monoclonal antibodies detected the S protein and immunoprecipitated the native form of the S protein. Indirect immunofluorescence and confocal analysis suggested strong reactivity of the antibodies towards the S protein of MERS-CoV virus infected Vero cells. Furthermore, the 506-2G10G5 monoclonal antibody significantly reduced plaque formation in MERS-CoV infected Vero cells compared to normal mouse IgG and 492-1G10E4E2. Thus, we successfully produced a monoclonal antibody directed against the RBD domain of the S protein which could be used in the development of diagnostics and therapeutic applications in the future.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.25 no.4
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• pp.312-320
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• 2022

Purpose: Screening serologic tests are important tools for the diagnosis of celiac disease (CD). Immunoglobulin (Ig)G anti-deamidated gliadin peptide (anti-DGP) is a relatively new autoantibody thought to have good diagnostic accuracy, comparable to that of anti-tissue transglutaminase (anti-tTG) antibody. Methods: Pediatric patients (n=86) with a clinical suspicion of CD were included. Duodenal biopsy, anti-tTG, and IgG anti-DGP antibody tests were performed. The patients were divided into CD and control groups based on the pathological evaluation of duodenal biopsies. The diagnostic accuracy of serological tests was determined. Results: IgA anti-tTG and IgG anti-DGP antibodies were positive in 86.3% and 95.4% of patients, respectively. The sensitivity, specificity, and diagnostic accuracy of the IgA anti-tTG test were 86.3%, 50.0%, and 68.6%, respectively, and those of the IgG anti-DGP test were 95.4%, 85.7%, and 90.7%, respectively. The area under the receiver operating characteristic (ROC) curve was 0.84 (95% confidence interval [CI], 0.74-0.91) for IgA anti-tTG test and 0.93 (95% CI, 0.86-0.97) for IgG anti-DGP test. The comparison of IgA anti-tTG and IgG anti-DGP ROC curves showed a higher sensitivity and specificity of the IgG anti-DGP test. Conclusion: IgG anti-DGP is a reliable serological test for CD diagnosis in children. High tTG and DGP titers in the serum are suggestive of severe duodenal atrophy. The combined use of IgA anti-tTG and IgG anti-DGP tests for the initial screening of CD can improve diagnostic sensitivity.

• Korean Journal Plant Pathology
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• v.14 no.3
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• pp.203-208
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• 1998

We isolated and characterized an antifungal peptide from the seeds of Phytolacca americana. Growth inhibition assay with Botrytis cinerea was used to screen inhibitory proteins from 60 different plant species. A 4 kDa antifungal peptide (Pa-AFP) inhibitory to hyphal growth of B. cinerea was found in the seeds of P. americana. The peptide Pa-AFP was purified to homogeneity by chromatographies of Sephadex G-50, DEAE-Sepharose, Sephacryl S-300, and C18 reverse-phase HPLC. Western blot analysis showed that a polyclonal antibody raised against the purified peptide cross-reacted with a 4 kDa protein in seeds but not in root and leaf tissues of P. americana. Pa-AFP inhibited the hyphal growth of Botrytis cinerea, Rihzoctonia solani, Fusarium oxysporum, and Magnaporthe grisea. Pa-AFP exhibited growth inhibition of Saccharomyces cerevisiae strain BWG7a, which was sensitive to osmotin.

• Biomolecules & Therapeutics
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• v.3 no.4
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• pp.266-272
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• 1995

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. To generate and characterize a moloclonal antibody against $\beta$-adrenergic receptor, a synthetic $\beta$2-adrenergic receptor peptide (Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-lle-Asp-Val-Leu) which may comprise part of $\beta$-adrenergic receptor ligand binding pocket was coupled to Keyhole Limpet Hemocyanin (KLH) and used as an immunogen. Male BALB/C mice were immunized with this antigen and the immunized spleen was fused with myeloma SP2/0-Ag14 cells to produce monoclonal antibodies. Two clones were obtained but one of monoclonal antibodies, mAb5G09, was used throughout in this study because the other clone, mAb5All showed weak immunoreactivity against KLH as well. The mouse monoclonal antibody mAb5G09 produced in this study showed immunoreactivity to peptide-KLH conjugates and also to human A43l cells and guinea pig lung $\beta$2-adrenergic receptor as revealed by ELISA and western blot. In the course of determination of the effects of mAb5G09 on $\beta$-receptor ligand binding, it was observed that mAb5G09 specifically bound $\beta$-adrenergic radioligand [$^3$H]dihydroalprenolol (DHA) with a dissociation constant (Kd) of 60 nM. The [$^3$H]DHA binding activity of mAb5G09 had characteristics of immunoglobulins and the binding activity was not observed in the control anti-KLH monoclonal antibody. The monoclonal antibody, mAb5G09 produced in this study may provide useful models for the study of the structure of receptor binding sites.