• IMMUNE NETWORK
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• 제12권1호
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• pp.33-39
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• 2012

Background: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). Methods: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reversetranscriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. Results: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. Conclusion: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.

• The Plant Pathology Journal
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• 제22권1호
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• pp.21-27
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• 2006

We immunized BALB/c mice with purified Cucumber green mottle mosaic virus isolate HY1 (CGMMV-HY1). Through the selection of positive clones that were grown on the HAT medium, four sensitive monoclonal clones (CG99-01, CG99-02, CG99-03, and CG99-04) were selected from 500 Hypoxanthine-guanine phosphoribosyltransferase positive hybridoma cells. Four sensitive clones of CGMMV-HYI were determined as IgM type of the subclass of mouse immunoglobulins Ig group. The titer of monoclonal antiserum against CGMMVHY1 was estimated 1:12,800 by the indirect ELISA. Although monoclonal antibodies (MAbs) from CG99-01 and from CG99-04 cross-reacted with Zucchini green mottle mosaic virus and Kyuri green mottle mosaic virus, MAb from the cell line CG99-03 was highly specific to CGMMV. No MAbs cross-reacted with Cucumber mosaic virus-Fny. Only CG99-04 reacted with Pepper mild mottle virus weakly and CG99-02 reacted with both CGMMV and KGMMV. CGMMV was detected from the rind of watermelon fruit by DAS-ELISA of CGMMV-HY1, but not from the flesh of watermelon. Average seed transmission rate of CGMMV in watermelon was $24\%$ from symptomatic watermelon collected from 5 regions of Gyeongnam province. CGMMV was detected by DAS-ELISA with specific MAb of CGMMVHY1 periodically from root stock, during the sequential process for nursery seedling in Haman. Necrotic spots on cotyledons of root stock seedling progressed to reveal the typical symptomatology on the primary leaves of scion upon grafting. Here, we have established MAb based ELISA system, which could accurately detect CGMMV from watermelon seeds, nursery seedlings, transplants and field samples from greenhouse or open out door field as well.

• 한국양식학회지
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• 제15권1호
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• pp.7-13
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• 2002

Since 1993, the White Spot Syndrome Virus (WSSV) disease occurred in China among cultured shrimps resulting in mass mortality. Epizootiological surveys undertaken during the outbreak period of 1993-1994 indicated that all stages of Penaeus chinensis, P. japonicus and P. monodon were infected. Consequent to the transport of contaminated shrimp seedlings and seawater, the disease spread all over the farms of China. The disease was more rapidly transmitted at temperatures above $25^{\circ}C$. Challenge experiments showed the causative agent was highly virulent. White spots appeared on the carapace of both span-taneous and experimentally infected shrimps. Moribund shrimps contained turbid hemolymph, hypertrophied Iymphoid organ and a necrotic mid-gut gland. Electron microscopy showed the presence of viral particles in the gills, stomach, lymphoid organ, and epidermal tissue of the infected shrimp. The visions were slightly ovoid with an envelope and averaged 350 $\times$ 150 nm; nucleocapsids measured 375 $\times$ 157 nm. With discontinuous sucrose gradient of 35, 50 and 60% (w/v), the virus was separated from hemolymph of the infected shrimp. The estimated molecular weight of genomic DNA was 237 Kb with EcoR I, 247 Kb with Hind III and 241kb with Pst I. A total of 9 hybridoma colones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with WSSV. The immunofluorescence assay of gill tissue showed that the MAbs reacted with diseased but not with healthy shrimp. The MAbs belonged to IgGl, IgG2b subclass and IgM class, all with kappa light Immune-electron-microscopy with colloidal gold marker showed the presence of 5 MAbs epitopes on the envelope and one on the capsid of the virus. Baculoviral mid-gut gland necrosis showed the specificity of the MAbs produced. For diagnosis 5 different methods were selected. Using Kimura primers for PCR, or MAbs for immunoblot, ELISA or FAT method, in situ hybridization was carried out to show the gene. All these methods detected WSSV in the organ samples of the diseased shrimp but not in healthy one.

• 한국가금학회지
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• 제15권3호
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• pp.207-210
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• 1988

뉴켓슬병바이러스(NDV)인 LaSota 주를 SPF 발육난의 요막강내에 증식시켜 순수 정제한 것을 BALB/c 흰쥐에 면역시킨 후 추출한 비강세포와 흰쥐 골수암세포와의 융합방법에 의하여 NDV에 특이하게 작용하는 단크론성항체(MCA)를 생산하는 3주의 Hybridoma틀 작성하였다. 이 3주의 MCA는 모두 IgG형에 속하였으며 흰쥐 복강 내에 접종하여 생산된 복수항체의 항체가는 간접형광항체법으로 $10^3$-$10^6$에 달하였고 약독 및 강독 NDV에 모두 동일한 수준으로 작용하였다. 중화능은 인정되지 않았고 3주중 1주만이 별구응집 억제능을 약하게 나타냈다. 이 MCA를 이용하여 간접형광항체법으로 인공 감염시킨 닭에서 NDV항원 검출을 시도한 결과 기관점막을 비롯한 각종 장기의 도말표본에서 접종 3일 후부터 뚜렷한 검출이 가능하였다.

• Archives of Pharmacal Research
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• 제29권12호
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• pp.1147-1153
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• 2006

Mizoribine (MZR) has been shown to possess immunosuppressive activity that selectively inhibits the proliferation of lymphocytes by interfering with inosine monophosphate dehydrogenase. The efficacy of MZR is not only in patients who have had renal transplantation, but also in patients with rheumatoid arthritis (RA), lupus nephritis, and primary nephritic syndrome. Because the exact mechanism of its immunosuppressive action is not clear, the object of this study was to examine the ability of MZR to regulate the antigen presenting cells (APCs), dendritic cells (DCs). In this work, we tested whether MZR ($1{\sim}10\;{\mu}g/mL$) could inhibit the cross-presentation of DCs. DC2.4 cells ($H-2K^{b}$) or bone marrow-derived DCs (BM-DCs) generated from BM cells of C57BL/6 mouse ($H-2K^{b}$) were cultured in the presence of MZR with OVA-microspheres, and the amount of OVA peptide-class I MHC complexes was measured by a T cell hybridoma, B3Z, that recognizes OVA (257-264 : SIINFEKL)-$H-2K^{b}$ complex and expresses-galactosidase. MZR profoundly inhibited the expression of SIINFEKL-$H-2K^{b}$ complexes. This inhibitory activity of MZR appeared to affect the phagocytic activity of DCs. MZR also decreased IL-2 production when we examined the effects of MZR on $CD4^{+}$ T cells. These results provide an understanding of the mechanism of immunosuppressive activity of MZR on the inhibition of MHC-restricted antigen presentation and phagocytic activity in relation to their actions on APCs.

• 한국작물학회지
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• 제36권6호
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• pp.506-512
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• 1991

IAA에 대한 단크론 항체를 생산하고 이를 이용하여 생체중의 내생 IAA를 정량분석하기 위해 ELISA를 개발하고 본법을 사용하여 담배 종자 발아중 내생 IAA함량을 정량분석하였다. 그 결과는 1. IAA에 대한 단크론 항체 생산 세포주 3가지를 선발 작성하였으며 이 세포주로부터 생산되는 항체는 모두 IgG$_1$ 타입의 면역 글로블린이었다. 2. 상기 항체를 사용하여 ELISA를 수행하여 표준곡선을 작성하였던 바 검출 한계는 1pmol이었으며 검출 범위는 500pmol이었다 3. 표준곡선으로부터 작성한 Scatchard plot에 의한 친화 상수와 결합상수는 6.7$\times$$10^{-10}$ L/M과 6$\times$$10^{-10}$ L/M이었다. 4. 여러가지 IAA유사물질과 교차반응에 의해서 본 mAb는 특이성이 매우 높고 RIA에 의해서 고역가임을 확인하였다 5. 발아중인 담배종자로부터 면역측정에 의해서 내생 IAA를 정량분석하였다 6. 상기의 결과에 따라서 본 mAb를 이용하여 생체중의 내생 IAA를 간편하게 정밀 분석할 수 있음을 확인하였다.

• BMB Reports
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• 제31권1호
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• pp.58-63
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• 1998

In this study, a mouse monoclonal antibody (mAb) against gp41(584-618), the immunodominant epitope protein, was generated. For this purpose, BALB/c mice were immunized with double branched multiple antigenic peptides derived from the HIV-1 gp41(584-618) sequence, and antibody-secreting hybridoma were produced by fusion of mice splenocytes with SP2/0 myeloma cells. One clone producing an antigen specific mAb, termed KI-41(isotype IgG1) was identified, whose specific reactivity against gp41(584-618) could be confirmed by ELISA and Western blot analysis. Epitope mapping revealed the recognition site of the mAb KI-41 to be located around the sequence RILAVERYLKDQQLLG, which comprises the N-terminal region within the immunized gp41(584-618) peptied. Since this mAb recognizes this specific epitope within the HIV-1 gp41 without any cross-reactivity to other immunodominant regions in the HIV-2 gp35, KI-41 will provide some alternative possibilities in further applications such as the development of indirect or competitive ELISA for specific antibody detection in HIV-1 infection or for other basic researches regarding the role and function of HIV-1 gp41.

• 대한수의학회지
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• 제44권2호
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• pp.259-268
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• 2004

Porcine circovirus type 2 (PCV-2) has been associated with various disease in pigs worldwide including postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). In this study, monoclonal antibodies (MAbs) against PCV were produced, characterized and applications of MAbs as diagnostic reagents were described. Spleen or lymph node cells from BALB/c mouse immunized respectively with PCV-1, PCV-2 or expressed PCV-2/ORF2 proteins in baculovirus were fused with SP2/0 myeloma cells using polyethylene glycol (PEG) and hybridoma cells producing PCV-1 or PCV-2-specific antibody were screened by an indirect immunofluorescence (IIF) test. A total of fifteen MAbs were produced against PCV. Six MAbs were PCV-1-specific and nine were PCV-2-specific. All PCV-1-specific MAbs reacted with only PCV-1 and all PCV-2-specific MAbs were reactive with only PCV-2 by IIF test. None of the MAbs was reactive with porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine rotavirus (PRV), and transmissible gastroenteritis virus (TGEV). Some PCV-2-specific MAbs recognized the PCV-2 infected porcine tissues by IIF or immunohistochemistry (IHC) assay. From this experiment, it was confirmed that MAbs produced in this study were PCV-specific and could be used as reliable diagnostic reagents for PCV-1/PCV-2 detection and differentiation.

• 한국해양바이오학회지
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• 제1권4호
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• pp.298-304
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• 2006

In this study, we establish a thin-layer chromatography (TLC) immunostaining method for detecting starfish gangliosides. A new monoclonal antibody (MAb) against AG-2, the major gangliosides molecular species of Acanthaster planci, was produced by fusing hybridoma with splenocytes immunized to liposomal AG-2. BALB/c male mice were injected with liposomal AG-2 antigen, and immunized. Their splenocytos were isolated and fused with hypoxanthine-aminopterine-thimidine (HAT)-sensitive mouse myeloma cells. Hybridomas producing MAb reactive to AG-2 were cloned using the limited dilution method. Established hybridomas were cultured in eRDF medium. Crude MAb produced from clone 8D4 was purified with a magnesium pyrophosphate column. Enzyme immunoassay and TLC immunostaining of AG-2 were performed using the purified MAb. Structurally related gangliosides did not cross-react with anti-AG-2 antibodies. The detection limit of TLC immunostaining was 50 ng of AG-2. The newly established immunostaining method was further developed for detecting AG-2 distribution and qualitative analysis in tissues and/or organs. Our results show that the majority of AG-2 is present in the stomach of male A. planci, while AG-2 is distributed not only in the stomach but also in the the pyloric caeca of female A. planci.

• 한국동물위생학회지
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• 제33권2호
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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.