Journal of Microbiology

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Immunological charaterization of monoclonal antibodies used in rapid influenza diagnostic test for detection of the 2009 pandemic influenza A(H1N1)pdm09 infection: Hwajung Yi , Mi-Seon Lee , Joo-Yeon Lee , Hae Kyung Lee , Chun Kang; J. Microbiol. 2015;53(2):166-175. Published online January 28, 2015; DOI: https://doi.org/10.1007/s12275-015-4642-2

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Since the 2009 pandemic, monoclonal antibodies (mAbs) for rapid influenza diagnostic tests (RIDT) have been developed for specific diagnostics of pandemic viral infection. Most of the mAbs were poorly characterized because of urgency during the pandemic. Further characterization of the mAbs for RIDTs would be beneficial for understanding the immunological properties of the pandemic virus and utilizing the mAbs for other research purposes. In this study, it was confirmed that two mAbs (I38 and D383) in an RIDT for H1N1pdm09 diagnostics were able to detect H1N1pdm09 virus through enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). Also, the two mAbs exhibited reactivity to hemagglutinins (HAs) of both the H1N1pdm09 and 1918 H1N1 viruses; therefore, the RIDT using the mAbs could detect HAs of H1N1pdm09 and also HAs of 1918 H1N1-like strains. In an extension to our previous study, the epitopes (Sa antigenic site and the interface area of F?and vestigial esterase subdomains on the HA1 domain of HA of H1N1pdm09) recognized by the mAbs were corroborated in depth by IFA with escape-mutants from the mAbs and mapping of the epitopes on the crystal structure of human H1N1 viral HAs. Collectively, these results imply that the mAbs for the RIDT may be suitable for use in studying the immunological properties of H1N1pdm09 viruses and that the Sa antigenic site and the interface area between F?and vestigial esterase subdomains on influenza viral HA recognized by the mAbs are immunologically conserved regions between H1N1pdm09 and 1918 H1N1.

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Antigenic diversity of H5 highly pathogenic avian influenza viruses of clade 2.3.4.4 isolated in Asia
Ayako Ohkawara, Masatoshi Okamatsu, Makoto Ozawa, Duc‐Huy Chu, Lam Thanh Nguyen, Takahiro Hiono, Keita Matsuno, Hiroshi Kida, Yoshihiro Sakoda
Microbiology and Immunology.2017; 61(5): 149. CrossRef

Fine Mapping of a Foot-and-Mouth Disease Virus Epitope Recognized by Serotype-Independent Monoclonal Antibody 4B2: Yongzhong Yu , Haiwei Wang , Lei Zhao , Chunyuan Zhang , Zhigang Jiang , Li Yu; J. Microbiol. 2011;49(1):94-101. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0134-1

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Abstract: VP2 is a structural protein of the foot-and-mouth disease virus (FMDV). In this study, a FMDV serotype-independent monoclonal antibody (MAb), 4B2, was generated. By screening a phage-displayed random 12-peptide library, we found positive phages displaying the consensus motif ETTXLE (X is any amino acid (aa)), which is highly homologous to 6ETTLLE11 at the N-terminus of the VP2 protein. Subsequently, a series of GST-fusion proteins expressing a truncated N-terminus of VP2 were examined by western blot analysis using the MAb 4B2. The results indicated that the motif 6ETTLLE11 of VP2 may be the minimal requirement of the epitope recognized by 4B2. Moreover, a 12-aa peptide 2KKTEETTLLEDR13 was shown to be the minimal unit of the epitope with maximal binding activity to 4B2. Alanine-scanning analysis demonstrated thatThr7, Thr8, and Leu10 are the functional residues of the 4B2 epitope Glu6 and Leu9 are required residues, and Glu11 plays a crucial role in the binding of MAb 4B2. The fine mapping of the epitope indicated that MAb 4B2 has the potential to be used in FMDV diagnosis.

Identification of a Novel Linear B-Cell Epitope in the M Protein of Avian Infectious Bronchitis Coronaviruses: Junji Xing , Shengwang Liu , Zongxi Han , Yuhao Shao , Huixin Li , Xiangang Kong; J. Microbiol. 2009;47(5):589-599. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0104-z

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Abstract: This report describes the identification of a novel linear B-cell epitope at the C-terminus of the membrane (M) protein of avian infectious bronchitis virus (IBV). A monoclonal antibody (MAb) (designated as 15E2) against the IBV M protein was prepared and a series of 14 partially-overlapping fragments of the IBV M gene were expressed with a GST tag. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA) and western blotting analysis using MAb 15E2 to identify the epitope. A linear motif, 199FATFVYAK206, which was located at the C-terminus of the M protein, was identified by MAb 15E2. ELISA and western blotting also showed that this epitope could be recognized by IBV-positive serum from chicken. Given that 15E2 showed reactivity with the 199FATFVYAK206 motif, expressed as a GST fusion protein, in both western blotting and in an ELISA, we proposed that this motif represented a linear B-cell epitope of the M protein. The 199FATFVYAK206 motif was the minimal requirement for reactivity as demonstrated by analysis of the reactivity of 15E2 with several truncated peptides that were derived from the motif. Alignment and comparison of the 15E2-defined epitope sequence with the sequences of other coronaviruses indicated that the epitope is well conserved among chicken and turkey coronaviruses. The identified epitope should be useful in clinical applications and as a tool for the further study of the structure and function of the M protein of IBV.

Generation and Characterization of a Monoclonal Antibody with Specificity for Mycoplasma arginini: Yeon Sung Son , Hyo Jeong Hong; J. Microbiol. 2007;45(6):547-552.; DOI: https://doi.org/2610 [pii]

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Abstract: Previously, we generated monoclonal antibodies (MAbs) that bound to the surface of human embryonic stem cells (hESCs) in an attempt to discover new hESC-specific surface markers. In this study, MAb 47-235 (IgG1, κ) was selected for further characterization. The MAb bound to the surface of undifferentiated hESCs but did not bind to mouse ESCs or mouse embryonic fibroblast cells in flow cytometric analysis. The antibody immunoprecipitated a 47 kDa protein from the lysates of cell surface-biotinylated hESCs. Identification of the protein by quadrupole time of flight tandem mass spectrometry revealed that 47-235 binds to Ag 243-5 protein of Mycoplasma arginini. BM-Cyclin treatment of the hESCs that reacted with 47-235 resulted in loss of mycoplasma DNA and the reactivity to 47-235. Nevertheless, the hESCs that were reactive to 47-235 maintained self-renewal and pluripotency and thus could be differentiated into three embryonic germ layers.

Guided Selection of Human Antibody Light Chains against TAG-72 Using a Phage Display Chain Shuffling Approach: Sang Jick Kim , Hyo Jeong Hong; J. Microbiol. 2007;45(6):572-577.; DOI: https://doi.org/2606 [pii]

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Abstract: To enhance therapeutic potential of murine monoclonal antibody, humanization by CDR grafting is usually used to reduce immunogenic mouse residues. Most humanized antibodies still have mouse residues critical for antigen binding, but the mouse residues may evoke immune responses in humans. Previously, we constructed a new humanized version (AKA) of mouse CC49 antibody specific for tumor-associated glycoprotein, TAG-72. In this study, to select a completely human antibody light chain against TAG-72, guided selection strategy using phage display was used. The heavy chain variable region (VH) of AKA was used to guide the selection of a human TAG-72-specific light chain variable region (VL) from a human VL repertoire constructed from human PBL. Most of the selected VLs were identified to be originated from the members of the human germline VK1 family, whereas the VL of AKA is more homologous to the VK4 family. Competition binding assay of the selected Fabs with mouse CC49 suggested that the epitopes of the Fabs overlap with that of CC49. In addition, they showed better antigen-binding affinity compared to parental AKA. The selected human VLs may be used to guide the selection of human VHs to get completely human anti-TAG72 antibody.

Expression and Purification of a Recombinant scFv towards the Exotoxin of the Pathogen, Burkholderia pseudomallei: Kue-Peng Lim , HongBin Li , Sheila Nathan; J. Microbiol. 2004;42(2):126-132.; DOI: https://doi.org/2034 [pii]

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Abstract: A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30^oC until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30^oC for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Top10F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, compared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.

Production of a monoclonal antibody and ultrastructure of the sporozoite of cryptosporidium parvum: Choi, Young Sook , Lee, Sung Tae , Cho, Myung Hwan; J. Microbiol. 1996;34(4):379-383.

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Abstract: Cryptosporidium Parvum causes a life-threatening diarrhea in acquired immunodeficiency syndrome (AIDS) patients. The sporozoite stage of C. parvum has been known to be a target in treating cryptosporidiosis in AIDS patients as it is an extracellular stage. A sporozoite was ultrastructurally observed. It has a crescent shape with a rounded posterior end and a tapering body. The compact nucleus was located at the posterior end. A monoclonal antibody was produced, which recognized a 43 kDa of sporozoite antigens in a western blot analysis and showed the surface labeling in immunofluorescence.

A detection method for vibrio vulnificus using monoclonal antibodies: Chung, Mi Sun , Rim, Bung Moo , Uhm, Tae Boong , Park, Moon Kook; J. Microbiol. 1997;35(2):87-91.

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Abstract: Monoclonal antibodies were prepared in order to an assay method for Vibrio vulnificus. Sixteen mouse ybridoma cell lines were established by immunization of whole cell antigen to BALB/c mice, fusion with SP2/O myeloma cells, and cloning. Most of them secreted IgM.lambda. antibodies. A sandwich enzyme-linked immunosorbent assay was developed with rabbit anti-V. vulnificus polyclonal antibodies as capture antibody, an IgM monoclonal antibody as detector antibody, and goat anti-mouse IgM-alkaline phosphatase conjugate as developer antibody. The range of detection was 10^4 to 10^7 V. vulnificus cells per microplate well. When four related Vibrio species were tested for cross-reactions, V. parahaemolyticus showed 3.5% reactively and V. carchariae, V. fluvialis, and V. furnisii showed negligibal (<1%) cross-reactivity.

Production and Characterization of Monoclonal Antibodies to Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein: Choi, Eui Yul , Ryu, Ji Yoon , Lee, Yoon , Ha, Sung Gil , Chung, So Young , Park, Sang Yeol , Nham, Sang Uk , Lee, Young Ik , Park, Jin Seu; J. Microbiol. 1998;36(1):59-65.

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Abstract: Monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoprotein gp 120(HIV-1 gp120) were produced and characterized. For immunogen recombinant gp120 polypeptide expressed in bacteria was prepared and injected into mice. From two fusion experiments, twenty hybridomas secreting monoclonal antibodies against the recombinant gp120 were initially screened by immunodot blot analysis. Among the antibodies, 15 of them showed strong reactivities with the recombinant protein expressed in bacteria in Western blot and thus it was tested if these could react with the recombinant protein expressed in insect cells. All of the 15 antibodies immunostained the protein band with varing degrees of reactivities. Next, we tested whether the antibodies recognize authentic gp120 protein expressed in mammalian cells. COS-1 cells were tranfected with the cDNA encoding gp120 protein, and the transiently ecpressed protein were analyzed with the mAbs by Western blot analysis and immunofluorescence microscopy. Six of the monoclonal antibodies reacted with the protein band of authentic gp120 expressed in mammalian cells in the Western blot, and five stained the cell periphery of the transfected COS-1 cells in immunofluorescence. The mAbs described in this study should prove to be useful tools for the biochemical, immunological and structural analysis of HIV-1 gp120 envelope glycoprotein.

Molecular Cloning and Characterization of cDNA Encoding Immunoglobulin Heavy and Light chain Variable Regions from Four Chicken Monoclonal Antibodies Specific to Surface Antigens of Intestinal Parasite, Eimeria acervulina: Ki Duk Song , Jae Yong Han , Wongi Min , Hyun S. Lillehoj , Sung Won Kim , Jin-Kyoo Kim; J. Microbiol. 2001;39(1):49-55.

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Abstract: We have developed four chicken hybridomas secreting monoclonal antibodies to induce a protective immune response against the chicken disease avian coccidiosis, caused by the intestinal parasite Eimeria acervulina. However, since the amount of antibodies secreted from these hybridomas is too low or sometimes they lost their ability to produce antibodies, the hybridoma method is not satisfactory in the production of large amounts of chicken monoclonal antibodies. To bypass these problems, we applied the antibody engineering technology using polymerase chain reaction. We cloned and determined the sequences of variable domains of the four chicken monoclonal antibodies, namely, 2-1, 5D11, 13C8 and 8C3. The sequences comparison to germline sequences showed that the gene conversion mechanism might contribute to developing diversification of heavy and l-light chains in chicken antibodies. Several pseudogene families regarded as donors in gene conversion were identified at each framework region and the complementarity determining region of l-light chains. In addition, as expected, numerous changes of nucleotide sequences such as nucleotide substitution, insertion and deletion were found predominantly in complementarity determining regions, which are likely to be somatic hypermutations as a result of affinity maturation in antibody-producing cells.

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