• 한국작물학회지
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• 제42권2호
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• pp.134-140
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• 1997

수도의 내염성 품종 안나프르나에 NaCl 50 mM을 처리하여 유도된 단백질의 분리정제를 수행하여 한 개의 새로운 단백질을 분리 정제하였다. 그 결과는 다음과 같다 1. 10일된 유묘에 50mM NaCl을 48시간 처리하여 유도된 단백질의 존재를 전기영동을 통하여 확인하였다. 2. FPLC를 이용한 DEAE와 phenyl column을 이용하여 순수한 정제가 가능하였고, 이 과정을 통하여 전기영동상에서 단일 밴드를 나타내는 하나의 단백질을 정제하였다. 3. 이 단백질의 분자량이 64,000이라는 것을 Se-phdex-G 100 column chromatography를 통하여 확인하였다. 4. 이 단백질에 대한 단일 항체를 조제하여 고역가 항체를 분비하는 hybridoma 세포주 3개를 작성하고 isotype등 특성을 조사하였다.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.226-229
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• 2012

Bacterial hemoglobin from Vitreoscilla (VHb) is recognized as a good fusion protein for the soluble expression of foreign protein. In this study, we generated a monoclonal antibody (MAb) against VHb for its detection. For the rapid screening of MAb, a protein chip technology based on the Alexa-488 (A488) dye labeling method was introduced. In order to fabricate the chip, the VHb protein was chemically coupled to the chip surface and then the culture supernatants of 84 hybridoma cell lines were spotted onto the VHb chip. The bound MAbs were measured by A488-modified anti-mouse IgG. A single spot (MAb A10) exhibited significantly high signal intensity. The immunoblot analysis evidenced that the MAb A10 can detect VHb-fused proteins with high specificity.

• 대한의생명과학회지
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• 제5권1호
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• pp.1-10
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• 1999

본 연구에서는 혈장이나 양수에 있는 $\alpha$-fetoprotein (AFP)을 인식 할 수 있는 모노클로날 항체를 제조하고, 모노클로날 항체를 이용한 효소면역분석법을 개발하고자 하였다. 양수로부터 얻은 AFP를 쥐에 주사한 후 비장을 분리하여 종양세포 (Sp2/O-Ag-14)와 융합하였고, 하이브리도마 기술을 이용하여 모노클로날 항체를 제조하였다. 모노클로날 항체를 클로닝하였으며, 생성된 항체를 MabF22로 명명하였고, IgG1 중사슬과 k 경사슬의 isotype을 나타냈다. 또한 immunoblotting 방법과 ELISA로 특이도를 조사한 결과 모노클로날 항체는 AFP와만 반응하였고, 결합 친화상수는 0.8$\times$$10^{-10}$M이었다. 두 종류의 효소면역분석법 -경쟁적 또는 비경쟁적 분석 -을 이용하여 항체의 효용성을 조사하였으며, 두 방법 모두 AFP와 농도에 비례하여 반응하였다. 따라서 본 연구에서 생산된 모노클로날 항체는 연구목적으로 뿐만 아니라 AFP 농도를 측정하기 위한 면역진단시약의 개발에도 유용할 것으로 생각된다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.133.1-133
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• 2003

Gummy stem blight, caused by Didymella bryoniae occurs exclusively on cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. Through this study, we optimized cultural conditions for mass-production of pycnidiospore by Metal Halide Lamp irradiation. In brief, the mycelial was cultured at $26^{\circ}C$ on PDA, for 2 days under the darkness, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$, a great number of pycnidia was simultaneously formed. Thus produced pycnidiospores were used as immunogen. From fusions of myeloma cell (v-653) with splenocytes from immunifed mice were car ried out. And, two hybridoma cell lines that recognized the immunogen Didymella bryoniae were obtained. One Monoclonal Antibody, Db1, recognized the supernatant and the other monoclonal antibody, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid two MAb Db1 and Db15, were immunotyped and identified as IgG1 and IgG2b, respectively. Titer of MAb Db1 and MAb Db15 was measured absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with Didymella bryoniae but none reacted with other of fungi and CMV, CGMMV Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{3}$ well by indirect ELISA characterization of the MAb Db1, Db15 antigen by heat and protease treatments show that the epitope recognized by the MAb Bb1, Db15 were a glycoprotein.

• The Plant Pathology Journal
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• 제19권5호
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• pp.260-265
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• 2003

Gummy stem blight caused by Didymella bryoniae occurs exclusively in cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. In this study, cultural conditions for the mass-production of pycnidiospore by Metal Halide (MH) lamp irradiation were maximized. The mycelia were cultured at $26^{\circ}C$ on PDA for 2 days under dark condition, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$. Results show that a great number of pycnidia were simultaneously formed. The pycnidiospores produced were then used as immunogen. Fusions of myeloma cell (v-653) with splenocytes from immunized mice were carried out. Two hybridoma cell lines that recognized the immunogen D. bryoniae were obtained. One monoclonal antibody (MAb), Dbl, recognized the supernatant while another MAb, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid of the two MAb, Dbl and Db15, the immunotypes of which were identified as IgG1 and IgG2b, respectively. Titers of MAb Dbl and MAb Db15 were measured and the absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with D. bryoniae but none reacted with other viral isolates, Cucumber mosaic virus and Cucumber green mottle mosaic virus. Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{-3}$/well by indirect ELISA. Characterization of the MAbs Dbl, Db15 antigen by heat and protease treatments, which suggests that the epitope recognized by these two MAbs was glycoprotein.

• 한국미생물·생명공학회지
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• 제14권3호
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• pp.219-223
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• 1986

사람 선유아세포 인터페론의 정제에 사용되는 단 clone성 항체생산 세포주를 조작하기 위하여 BA-LB/C mouse의 복강과 꼬리정맥을 통하여 HuIFN-$\beta$를 면역화시키고 그 비장세포(spleen cells) 와 NS-O 세포주를 세포융합 시켰다. 융합된 1300 hybrids를 ELISA방법으로 선별하고 soft agarose 방법과 limiting dilution방법으로 subcloning하여 높은 항체를 생성하는 것으로 판명된 11 hybrids를 재선별 하였다. 재선별된 11 hybrids 각각의 항체형 (Ig type)을 조사하고 최종 Protein A-sepharose와 친화성이 높은 IgG 2a/형의 clone # 4-1-19와 clone # 551-4-1을 선별하여 배양된 세포를 각각 nude(nu/nu) mouse 및 BALB/c mouse 복강에 접종배양 하였다. 이들 mouse복강액으로 부터 얻은 ascites fluid를 protein A-sepharose를 이용한 affinity column분획으로 항체를 정제하였으며 ascites fluid $m{\ell}$당 약 4mg의 정제된 항체를 얻을 수 있었고 SDS-polyacrylamide gel상에서 전기영동 시킨 결과 분자량 14-16만 dalton으로 추정되는 항체를 확인할 수 있었다.

• 한국식품조리과학회지
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• 제27권4호
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• pp.67-73
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• 2011

This study was performed to assess the neuroprotective effects of methanolic extracts from sweet persimmon peel (PPE) against glutamate-induced cytotoxicity in hybridoma N18-RE-105 cells. The neuroprotective effects of PPE in N18-RE-105 cells were measured using the MTT reduction assay, LDH release assay, and phase-contrast microscopy. The results of the MTT reduction assay showed that treating cells with 500 ${\mu}g/ml$ PPE resulted in cell viability of 66.9%. Additionally, the morphological changes and the results of the LDH release assay showed that glutamate-induced damage to nerve cells was strongly inhibited by PPE. GSH content of N18-RE-105 cells was 3.5 ${\mu}M$ compared to that of the control, whereas pretreatment with 500 ${\mu}g/ml$ PPE increased GSH content by 4.7 ${\mu}M$. PPE was fractionated with hexane, and that layer had the highest neuroprotective effects in glutamate-stressed N18-RE-105 cells. In conclusion, our data showed that glutamate potentiated the effects of N18-RE-105 cell death by a mechanism involving oxidative stress. Therefore, PPE may be a potential candidate for prevention and therapy of neurodegenerative diseases.

• 대한미생물학회지
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• 제21권3호
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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.

• Journal of Periodontal and Implant Science
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• 제24권3호
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• pp.472-482
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• 1994

Cytokines expressed specifically in leukocytes subsets and in activated cells, which are involved in chemotaxis and activation of leukocytes, are recently defined as chemokines. Macrophage inflammatory $protein-1{\alpha}(MIP-1{\alpha})$ and $MIP-1{\beta}$ are members of C-C chemokine subfamily which produces wide immunomodulatory, proinflammatory, and hematopoietic modulatory actions. We have studied their gene expression by using Northern blot analysis in various blood cells such as cytolytic T lymphocyte(CTL), helper T lymphocyte(HTL), macrophage, and B lymphocyte. Resting CTL line CTLL-R8 expressed $MIP-1{\alpha}$ mRNA which was downregulated by ConA stimulation. Both of resting and ConA stimulated HTL line Hut78 and Jurkat did not express $MIP-1{\alpha}$ mRNA. There was detectable $MIP-1{\alpha}$ transcript in HTL hybridoma 2B4.11 which was a little upstimulated by ConA stimulation. B cell line 230, and macrophage cell line RAW264.7 and WR19M.1 showed distinct $MIP-1{\alpha}$ message which were induced after LPS stimulation. Expression pattern of $MIP-1{\beta}$ in all cell lines or cell were almost identical to that of $MIP-1{\alpha}$. Also strategies employed to identify and characterize the biological functions was preceded by receptor cloning to trace the shorcut to the final goal of cytokine research. For the cloning of $MIP-1{\alpha}$ receptor(R), we used synthetic oligonucleotides of transmembrane(T) conserved sequences of already cloned human(h) IL-8-R, and performed reverse transcription-polymerase chain reaction(RT-PCR) amplification using murine(m) macrophage cell line mRNA. Among 5RT-PCR products, we isolated a homologous cDNA with hIL-8-R which were shown to be putative mIL-8-R cDNA.

• 미생물학회지
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• 제51권2호
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• pp.115-125
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• 2015

단일클론항체 AP64 IgM은 인간의 급성 비임파성 골수암(ANLL) 세포주 HL60에 결합하며 쥐의 ANLL 세포에도 교차결합(cross-react)한다. 또한 complement에 의해 매개되어지면 골수암 억제효과를 나타낸다. 본 연구에서는 RT-PCR에 의해 AP64 IgM을 분비하는 하이브리도마의 $V_H$ 및 $V_L$ cDNA로부터 유래된 재조합 single-chain variable domain fragment (ScFv)를 제조하였다. $V_H$ 및 $V_L$은 15개 아미노산으로 구성된 linker $(G_4S)_3$으로 연결되었다. 재조합 ScFv는 Escherichia coli BMH 71-18에서 single polypeptide chain으로 발현되었다. Periplasmic extract를 $Ni^+$-NTA-agarose affinity column에 가하여 발현된 재조합 ScFv를 정제하였으며 westernblot으로 정제된 단백질을 탐지하였다. 정제된 재조합 ScFv는 AP64 IgM 모항체가 탐지하는 항원과 같은 HL60 세포의 표면항원(약 30 kDa)을 인지하였다. 그러나 HL60의 표면항원에 대한 ScFv의 결합력은 AP64 IgM 모항체보다 낮아서 추후 이에 대한 개선이 필요하다. 종합하여 볼 때 HL60 세포주에 특이적인 재조합 ScFv는 진단 또는 치료목적으로 유용한 생물학적 제재가 될 수 있을 것이다.