• Microbiology and Biotechnology Letters
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• v.25 no.4
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• pp.414-419
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• 1997

In order to develop an enzyme-linked immunosorbent assay (ELISA) for deoxynivalenol (DON) in com, we produced a specific monocl- onal antibody and established ELISA conditions. After the spleen cells from mice immunized with DON-bovine serum albumin conjugate were fused with S$_{p}$2/0 myeloma cells, a hybridoma cell 3G7 producing anti-DON antibody was screened by ELISA. From the standard curve of competitive direct ELISA (cdELISA) using 3G7 monoclonal antibody and DON-HRP conjugate, the detection range of DON showed 3-3,000 ng/ml (ppb). The monoclonal antibody showed some cross-reactivities against DON analogues such as 15 acetyl-DON (110%), nivalenol (5.0%), 3 acetyl-DON (1.7%), fusarenon-x (0.72%), and T-2 (0.59%). When the cdELISA was applied to the spiked coms after extracting with 60% methanol and diluting 5- fold with washing buffer, the assay recoveries of DON were 313, 163, 106, and 88.9% (av., 168%) in the levels of 200, 600, 2,000, and 6,000ng/g, respectively. For the quantitation of DON in coms, 30 samples kept under two different storage conditions of cold and room temperature were assayed by cdELISA. The mean detection concentrations were 595 (detection range, 0-2,750) and 2,448 (detection range, 0-4,500) ppb, respectively.

• Journal of fish pathology
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• v.30 no.2
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• pp.149-154
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• 2017

Mouse monoclonal antibodies (MAbs) were produced by using viral hemorrhagic septicemia virus (VHSV, genotype IVa) as an immunogen, isolated from diseased olive flounder (Paralichthys olivaceus). Four hybridoma clones secreting MAbs against VHSV were established. The MAbs were recognized the nucleoprotein (MAb 4), phosphoprotein (MAb 1) and matrix protein (MAbs 2 and 3) of VHSV by western blot analysis. Among them, the MAbs 1 and 4 strongly reacted with the VHSV-infected FHM cells, but not normal FHM cells. In enzyme linked immunosorbent assay, the four MAbs reacted with the VHSV, but not different six fish viruses (infectious hematopoietic necrosis virus, hirame rhabdovirus, spring viraemia of carp virus, infectious pancreatic necrosis virus, marine birnavirus and nervous necrosis virus). These results indicate that the MAbs are useful for diagnosis of VHSV infection.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2000.05a
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• pp.446-446
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• 2000

Specific polyclonal and/or monoclonal antibodies (MAbs) to immunoglobulins (Igs) and their subunits have proved to be valuable tools in immunological research and in immunological assays. In this study, we developed and characterized MAbs against flounder serum Igs. To obtain the pure flounder serum Igs, mouse IgG (mIgG) was immunized to flounder. Flounder Igs were purified by using mIgG-agarose affinity column chromatography. The structure of purified flounder Ig was observed, on denatured SDS-PAGE, to be composed of two heavy chains (77 and 72 kd) and two light chains (28 and 26 kd). MAbs were produced by fusion of myeloma cells (SP2/0) with Balb/c mouse spleen cells previously primed with the flounder Igs. Finally, three hybridoma clones, FIM 511, FIM 519 and FIM 562 were established to recognize both 2 heavy chains, 26 kd of light chain and 28 kd of light chain, respectively. On the other hand, the flounder immune sera collected on the weekly basis were tested on ELISA and immunoblot analysis whether boosting effect is present in flounder humoral immune system. As a result, the secondary immune response in flounder was ascertained on ELISA, but not on immunoblot analysis. Further, we observed an alteration of serum protein levels following immunization. Our MAbs and basic information on flounder humoral immune system obtained in this study will be helpful to control and monitor the efficiency of fish vaccines and therapeutic process of flounder diseases.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.71-80
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• 1996

The envelope of the rnannnalian oocyte plays crucial roles in sperm-oocyte interactions by providing sperm receptors, inducing acrosome reaction and preventing polyspermy. Understanding of properties of the zona pellucida (ZP) is essential for the artificial control of fertility in mammals. This study was carried out to produce and characterize monoclonal antibodies(MAbs) to porcine ZP proteins. Approximately 8,000 ZPs were obtained from follicular oocytes and dissolved in 40$\mu$l of double distilled water. Following immunization through foot-pad injections of Balb /c mice with a ZP solution, the popliteal lymph nodes were recovered at 2 weeks after the last injection. Hybridoma cell lines were established by fusing lymph node cells with P3X63 myeloma cells through selection using HAT medium and screening by immunofluorescence(IF) microscopy on the isolated ZP. Secreted MAbs were found to consist k chains and different heavy chains as evidenced by isotyping. Some of the MAbs demonstrated high specificity to the ZP in IF. The Mabs also showed positive cross reactivity with hamster and mouse eggs, while negative with bovine eggs. The results implicate that the MAbs can be used not only for identification of functional regions of the ZP, but also for elucidation of mechanisms involved in fertilization of mammals. The MAbs will provide basic information on biochemical anatomy of the ZP as well as can be candidates for the future contraceptive vaccines.

• Korean Journal of Veterinary Research
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• v.39 no.6
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• pp.1112-1118
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• 1999

Galectin-3 plays an important role in cell development, differentiation and cancer metastasis, including cell-cell/extracellular matrix (ECM) interactions and is supposed to have an effect of apoptosis on T-cells in thymic clonal selection. In this study, to know the effect of galectin-3 on thymocyte development, we used recombinant human galectin-3 (rHgal-3) from Escherichia coli, JM105, which was inserted with human gal-3 gene-transformed plasmid vector (prGal-3) to express human galectin-3. Expressed rHgal-3 was confirmed by western blot using the culture supernant of hybridoma (M3/38) producing monoclonal antibody to human galectin-3. Sepharose gel affinity chromatography was used to purify the expressed rHgal-3. Thymocytes and hepatocytes from 6-week-old male BALB/c mice were incubated with rHgal-3 and showed marked increase of apoptotic population on analysis using flow cytometry with 7-AAD in a dosedependent manner. However, rHgal-3 failed to induce apoptosis on hepatocytes. Interestingly, this apoptotic effect was not inhibited by lactose, a specific lectin domain inhibitor. From these results, we concluded that extrinsic -3 induces apoptosis on mouse thymocytes, and galectin-3 may have an apoptotic effect on T-cells in thymic clonal selection.

• Korean Journal of Microbiology
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• v.33 no.2
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• pp.97-104
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• 1997

Herpes simpex virus type 2 (HSV-2) infects the genital and oral mucosae of human and other animals. HSV-2 infection is a widespread health problem causing various clinical syndromes including oral, genital, and ocular lesions, viral encephalitis, and recurrent diseases. Hybridorna cell lines secreting a monoclonal antibody (mAb) against the HSV-2 were produced by fusing spleen cells of HSV-2-immunized mice with Sp2/0-AgI4 myeloma cells. One hybridoma cell line was established and its monoclonal C-2, IgM, recognized the antigens of 134, 86, and 43 kDa in western blot analysis. In SDS-P AGE analysis of HSV -2 antigens, 25 bands were separated between 3D kDa and 159 kDa. In indirect immunofluorescent assay, mAbs exhibited binding to the virus antigen expressed on Vero cell infected with HSV-2.

• Journal of fish pathology
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• v.32 no.2
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• pp.59-67
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• 2019

We developed and subsequently characterized mouse antibodies (MAbs) against viral hemorrhagic septicemia virus (VHSV, genotype IVa), the causative agent of VHS. Five hybridoma clones secreting MAbs against VHSV were established. The MAbs recognized the glycoprotein (MAbs 2C10, 18H4, 23H6, and 30B7) and nucleocapsid protein (15E10) of VHSV by western blot analysis. All five MAbs reacted with VHSV-infected cells and tissue homogenates of VHSV-infected olive flounder (Paralichthys olivaceus) by western blot analysis. Whereas, no reactivity was observed in normal cells and tissue homogenates of normal olive flounder. Moreover, these MAbs reacted with VHSV, but did not react with other fish viruses (infectious hematopoietic necrosis virus, hirame rhabdovirus, spring viraemia of carp virus, infectious pancreatic necrosis virus, marine birnavirus, and nervous necrosis virus) by enzyme linked immunosorbent assay (ELISA). These results indicate that the MAbs are specific to VHSV and can be of value in VHSV detection.

• Biomedical Science Letters
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• v.5 no.1
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• pp.1-10
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• 1999

This study was attempted to generate a monoclonal antibody against human $\alpha$-fetoprotein (AFP) and to produce an immunoassay, recognizing AFP in plasma and amniotic fluid. AFP was purified from human amniotic fluid and used to immunize mice. Spleens were taken from the mice and the cells were fused with mouse myeloma cells (Sp2/0-Ag-14) for the production of monoclonal antibodies by employing the hybridoma technology. As a result, a hybridoma cell line producing anti-AFP monoclonal antibody was cloned out and designated as MabF22. From isotyping analysis, it was found that monoclonal antibody MabF22 was IgG type with IgG1 heavy chain and k light chain. The binding specificity of MabF22 was analyzed by immunoblotting as well as by ELISA. MabF22 was highly specific, reacting with only AFP-containing samples. The binding affinity was determined by ELISA (free-capture mode) and Scatchard analysis. As a result, the value of Kd was 0.8$\times$10$^{-10}$M. The validity of the MabF22 for AFP assay was examined by two kinds of ELISAs, i.e., non-competitive and competitive ELISA. Both assays revealed that MabF22 reacted well with AFP in sample in a concentration-dependent manner. Standard curve and antibody titration curve were obtained by using purified AFP and MabF22. These results indicate that the monoclonal antibody produced in this study would be useful not only for research purposes but also for further development of immune-diagnostic kit for the measurement of AEP concentration.

• Microbiology and Biotechnology Letters
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• v.14 no.3
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• pp.219-223
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• 1986

In order to preparr the hybridoma cells which produce a monoclonal antibody to human fibroblast interferon(Hu IFN-$\beta$), spleen cells from BALB/cmice immunized with the purified Hu IFN-$\beta$ were fused with NS-O cells, a myeloma cell line. Forty hybrids with high titer among 1300 hybrids Isolated by an ELISA screening method were subcloned using the soft agarose cloning and limiting dilution methods, and 11 hybrids were selected. As a result of iso-typing the hybrids using the mouse monoclonal typing kit, two hybridomas were found to produce 1gG 2a type of monoclonal an-tibodies. The ascites fluid from nude mice inoculated intraperitoneally with the above hybridomas was removed and purified using a protein A-Sepharose CL-4B. Monoclonal antibody was proven to have only the heavy and light chains on SDS-polyacrylamide gel electrophoresis.

• Korean Journal of Veterinary Service
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• v.33 no.2
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• pp.105-111
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• 2010

Rabies virus which belongs to the genus Lyssavirus of the family Rhabdoviridae is known as a highly neurotropic virus and causes fatal encephalitis accompanied by severe neurological symptoms in almost all mammals, including humans. In this study, monoclonal antibodies (MAbs) against rabies virus were produced, characterized and applications of MAbs as diagnostic reagents were assessed Spleen and inguinal lymph node cells from Balb/c mouse immunized with purified rabies virus were fused with SP2/O myeloma cells using polyethylene glycol (PEG) and hybridoma cells producing rabies virus-specific MAbs were screened by an indirect fluorescent antibody test. A total of ten MAbs were produced against rabies virus. The protein specificity and neutralizing activity of MAbs were determined by Western blot analysis and fluorescent antibody virus neutralization test, respectively. As a result, two MAbs, 5G3 and 6H4 had specificity for nucleoprotein (N protein) and two other MAbs, 5B1 and 5C1 had neutralizing activity for rabies virus. Some MAbs recognized the rabies virus-infected bovine brain stem cells by immunohistochemistry (IHC) assay. In conclusion, it was confirmed that MAbs produced in this study were rabies virusspecific and could be used as reliable diagnostic reagents for the detection of rabies virus.