• 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.

• IMMUNE NETWORK
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• v.1 no.2
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• pp.126-134
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• 2001

Background : Paeonia japonica Miyabe is a medicinal plant which has been widely used as a component of blood-building decoctions (Chinese medicinal concept : Bu-Xie). The immunopharmacological characteristics of the extract of Paeonia japonica (PJ) were investigated. Methods : The effects of fractions of PJ extract on lymphocyte proliferation were measured by $H^3$-thymidine incorporation assay. The proliferated lymphocyte subsets were analyzed in flow cytometry. The subset cell populations of spleen cells were separated by magnetic cell separation system, and their proliferation by the extract were investigated. The effect of the extract on antibody production was determined in mice challenged with sheep red blood cells (SRBC) using hemolytic plaque forming cell assay. Results : Spleen cells were proliferated by water extract of PJ. Polysaccharide fraction (PJ-P) of the extract was most active in the proliferation. It was found in flow cytometry that the lymphocyte subset proliferated by PJ-P was B cell population. Among the separated subset cell populations, T cell-depleted cell population and macrophage-depleted cell population were most proliferated by PJ-P. However, positively selected populations of B cells and T cells were not proliferated by PJ-P. These results indicate that B cell proliferation by PJ-P may require the assistance of macrophages or T cells. These results suggest that firstly PJ-P may stimulate macrophages or T cells, and then B cells are activated. The number of antibody-secreting cells was increased by administration of PJ-P in mice immunized with SRBC as a T-dependent antigen. Conclusion : These results suggest that macrophages and accessory cells are directly activated by PJ-P and then helper T cells and B cells are indirectly activated. As the results, immune responses might be coordinately improved. In conclusion, PJ-P, a polysaccharide of P. japonica, may be a characteristic immunostimulator, which is analogous to polysaccharides such as lentinan, PSK and ginsan.

• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1176-1184
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• 2013

Anti-hepatocyte growth factor (anti-HGF) monoclonal antibodies (mAbs) are potential therapeutics against various cancers. Screening for high-producer clones is a time-consuming and complex process and is a major hurdle in the development of therapeutic mAbs. Here, we describe an efficient approach that allows the selection of high-producer Chinese hamster ovary (CHO) cell lines producing the novel anti-HGF mAb SFN68, which was generated previously by immunizing HGF bound to its receptor c-Met. We selected an SFN68-producing parental cell line via transfection of the dihydrofolate reductase-deficient CHO cell line DG44, which was preadapted to serum-free suspension culture, with an SFN68-expression vector. Subsequent gene amplification via multiple passages of the parental cell line in a methotrexate-containing medium over 4 weeks, followed by clonal isolation, enabled us to isolate two cell lines, 2F7 and 2H4, with 3-fold higher specific productivity. We also screened 72 different media formulated with diverse feed and basal media to develop a suboptimized medium. In the established suboptimized medium, the highest anti-HGF mAb yields of the 2F7 and 2H4 clones were 842 and 861 mg/l, respectively, which were about 10.5-fold higher than that of the parental cell line in a non-optimized basal medium. The selected CHO cell lines secreting high titers of SFN68 would be useful for the production of sufficient amounts of antibodies for efficacy evaluation in preclinical and early clinical studies.

• The Journal of the Korean Society for Microbiology
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• v.21 no.3
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• pp.399-406
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• 1986

This study was performed to establish human plasmacytoma cell line as the partner cells for producing human hybridoma. Bone marrow cells from a multiple myeloma patient from Seoul National University Hospital, Korea were cultured and established as the cell line, named as HMC-BM4. HMC-BM4 cells were cultivated in RPMI 1640 media containing 8-azaguanine(8-AG; gradually increasing concentration from $1\;{\mu}g/ml$ to $20\;{\mu}g/ml$). 8-AG resistant cells were collected and cloned by limiting dilution. Each clone was divided and tested to die in hypoxanthine, aminopterine and thymidine (HAT) selection media. Finally one clone was selected and named as HMC-AR, which was sensitive to HAT selection media. HMC-AR cells showed typical morphology of plamacytoma in Wright staining. No cell formed the rosette with sheep erythrocytes. Surface membrane $\mu$ heavy chain was detected in 20% of HMC-AR cells and cytoplasmic $\mu$ heavy chain in 90% of them by direct immunofluorescent staining. Ia-like antigen was found in 90% of HMC-AR cells by indirect immunofluorescent staining using anti-Ia-like antigen monoclonal antibody, 1BD9-2. And about $1.0\;{\mu}g/ml$ of human $\mu$ heavy chain was detected in the 3-day culture supernatant of HMC-AR cells. 88% of cells contained 46 chromosomes. Mycoplasma was not detected in HMC-AR cells by Hoechst 33258 staining. This cell line would be used for making hybridomas secreting human monoclonal antibody.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.1
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• pp.1-7
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• 2002

To study the increased resistance in fish against Vibrio vulnificus known as an important agent of vibrio septicemia in human, we analyzed specific and nonspecific immune response in flounder after administration of V. vulnificus bacterins by oral route. It contained the comparison of antibody concentrations in the sera of flounder after oral administration by two different protocols with uncoated heat killed bacterin of V vulnificus (UHKB, 20 mg/kg body weight), i.e., 4 weeks continuously (group 4W) and taking 2 weeks resting period between the 1st and last week of administration (group 1-2-1W). Even though, 1-2-1W group showed significantly increased level of specific antibody in serum, it did not reach to that of 4W group. Certainly, flounder vaccinated twice a week for four weeks (20 mg/kg b.w.) showed increased concentration of specific antibody against V. vulnificus at week 2 after last administration by oral route and maintained throughout the experimental period. It also was confirmed by the increased numbers of specific antibody secreting cells (SASC) in the leukocytes isolated from the splenocytes of the flounder of 4W group at week 1 after last administration until the end of experimental period. However, enteric, acid-resistant film coated heat killed bacterin (ECHB) did not show both greater immune reaction for antibody production and faster elimination of a challenge dose of V. vulnificus compared with those of the UHKB. These results suggested that UHKB administered by oral route was very effective method to prevent the contamination of V vulnificus in flounder, and did not show the increased antigenicity by coating the surface with acid-resistant film.

• IMMUNE NETWORK
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• v.8 no.1
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• pp.7-12
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• 2008

Background: Members belonging to the interferon-lambda (IFN-${\lambda}$) family exert protective action against viral infection; however, the mechanisms of their action have remained elusive. To study IFN-${\lambda}$ biology, such as endocytosis of IFN-${\lambda}$, we produced monoclonal antibodies (Abs) against human IFN-${\lambda}$ and examined their usefulness. Methods: We purified recombinant human IFN-${\lambda}$1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-${\lambda}$1-immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-${\lambda}$1, we performed RT-PCR for the MxA transcript. In order to study the binding activity of IFN-${\lambda}$ and the monoclonal Ab complex on HepG2 cells, we labeled the monoclonal Ab with rhodamine and determined the fluorescence intensity. Results: Four hybridoma clones secreting Abs specific to IFN-${\lambda}$1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-${\lambda}$1 in the denatured form as well as in the native form. Abs produced by HL1, HL3, and HL4 did not neutralize the induction of the MxA gene by IFN-${\lambda}$1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-${\lambda}$ complex on HepG2 cells. Conclusion: Monoclonal Abs against IFN-${\lambda}$1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-${\lambda}$.

• The Journal of the Korean Society for Microbiology
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• v.21 no.1
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• pp.151-161
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• 1986

In order to develop sensitive and sepcific assay methods for E. coli heat labile enterotoxin(LT) hybridoma cell lines secreting LT specific monoclonal antibody were obtained. LT was purified from cell lysate of E. coli O15H11. The steps included disruption of bacteria by French pressure, DEAE Sephacel ion exchange chromatography, Sephadex G200 gel filtration, and second DEAE Sephacel ion exchange chromatography, successively. Spleen cells from Balb/c mice immunized with the purified LT and $HGPRT^{(-)}$ plasmacytomas, $P3{\times}63Ag8.V653$ were mixed and fused by 50% (w/v) PEG. Hybrid cells were grown in 308 wells out of 360 wells, and 13 wells out of them secreted antibodies reacting to LT. Among these hybridoma cell 1G8-1D1 cell line was selected since it had produced high-titered monoclonal antibody continuously. By using culture supernatant and ascites from 1G8-1D1 cells the monoclonal antibody was characterized, and an assay system for detecting enterotoxigenic E. coli was established by double sandwich enzyme-linked immunosorbent assay (ELISA). The following results were obtained. 1. Antibody titers of culture supernatant and ascites from 1G8-1D1 hybridoma cells were 512, and 102, 400, respectively by GM1-ELISA and its immunoglobulin class was IgM. 2. The maximum absorption ratio of 1G8-1D1 cell culture supernatant to LT was 90% at $300\;{\mu}g/ml$ of LT concentration. LT concentration shown at 50% absorption ratio was $103.45{\mu}g$ and the absorption ratio was decreased with tile reduction of LT concentration. This result suggests that monoclonal antibody from 1G8-1D1 hybridoma cell bound with LT specifically. 3. The reactivities of 1G8-1D1 cell culture supernatant to LT and V. cholerae enterotoxin(CT) were 0.886 and 0.142(O.D. at 492nm) measured by the GM1-ELISA, indicating 1G8-1D1 monoclonal antibody reacted specifically with LT but not with CT. 4. The addition of 0.1ml of ascites to 0.6mg and 0.12mg of LT decreased the vascular permeability factor to 41% and 44% respectively, but it did not completely neutralize LT. 5. By double sandwich ELISA using monoclonal antibody, as little as 75ng of the purified LT per ml could be detected. 6. The results by assay of detecting LT in culture supernatants of 14 wild strains E. coli isolated from diarrhea patients by the double sandwich ELISA were almost the same level as those by reverse passive latex agglutination.

• Parasites, Hosts and Diseases
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• v.57 no.1
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• pp.33-38
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• 2019

Trichomoniasis is a common sexually transmitted infection caused by Trichomonas vaginalis, which actually does not exist a vaccine for control or prevention. Thus, the identification of new and potent immunogens in T. vaginalis, which can contribute to the development of a vaccine against this parasite, is necessary. Therefore, the aim of this work was to evaluate the potential of a recombinant Transient Receptor Potential-like channel of T. vaginalis (TvTRPV), as a promising immunogen in BALB/c mice. First, TvTRPV was cloned and expressed as a recombinant protein in Escherichia coli BL21 cells and purified by nickel affinity. Next, BALB/c mice were immunized and the antibody levels in mice serum and cytokines from the supernatant of macrophages and from co-culture systems were evaluated. Recombinant TvTRPV triggered high levels of specific total IgG in sera from the immunized mice. Also, a statistically significant increase of cytokines: $IL-1{\beta}$, IL-6, and $TNF-{\alpha}$ after stimulation with the corresponding antigens in vitro, was identified. Moreover, co-cultures using $CD4^+$ T cells from immunized mice were able to identify higher levels of IL-10 and $IFN-{\gamma}$. These results were useful to validate the immunogenicity of TvTRPV in BALB/c mice, where IL-10-$IFN-{\gamma}$-secreting cells could play a role in infection control, supporting the potential of TvTRPV as a promising target for vaccine against T. vaginalis.

• 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.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.2031-2033
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• 2016

Background: Symptomatic multiple myeloma (MM) is an acquired B-cell malignant proliferation of antibody secreting plasma cells, characterized by end organ damage due to monoclonal immunoglobulin secretion. The aim of this study wa to determine the stage stratification according to an international scoring system in adult Pakistani MM patients at presentation. Materials and Methods: This single centre retrospective study extendedfrom January 2012 to December 2015. Data were retrieved from the departmental maintained records. Results: A total of 39 patients were diagnosed at our center with MM during the period of the study, 25 males and 14 females. Age ranged between 36 and 81 with a mean of $54.5{\pm}14.8$ and a median of 57 years. Common presenting complaints included fatigue (80.9%), backache (79.3%) and bone pain (66.2%). Overall, 9 patients were in ISS stage I (23%), 12 were in stage II (30.7%) and 18 were in stage III (46.1%). Out of the total, 29 (74.3%) had kappa immunoglobulin andthe remaining 10 (25.6%) had lambda type myelomas. IgG myeloma was commonest, seen in 26 (66.6%) followed by IgA in 11 (28.2%) with non secretory myeloma in one (2.5%) and light chain disease also in one patient (2.5%). Conclusions: MM in Pakistani patients is seen in a relatively young population with male predominance. Primarily patients are symptomatic and risk stratification revealed a predominance of advanced stage III disease in our setting.