• 공업화학
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• 제30권4호
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• pp.429-434
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• 2019

본 논문에서는 수용성의 CdSe/ZnS 양자점을 합성하고 이에 항체기능성을 도입하여 lateral flow immunoassay (LFIA) 플랫폼에 융합하여 폐암 질병진단에 활용 가능한 단백질 바이오마커[예: 인간 혈청 아밀로이드 A-1 (hSAA1)]의 농도 분석에 적용하고자 한다. 면역분석법 센서 스트립은 니트로셀룰로오즈 막에 테스트라인과 대조라인으로 각각 항hSAA1 단일클론항체(10G1)(anti-hSAA1)와 항chicken IgY (anti-chicken IgY)를 스프레이하여 제작하였다. 이와 함께, 유기상에서 합성된 CdSe/ZnS 양자점은 카르복실기로 변형된 알케인티올기를 이용한 리간드 교환방법으로 수용성으로 전환하였으며, 이에 타겟 단백질인 hSAA1에 특이적으로 결합 가능한 항체인 항hSAA1 단일클론항체(14F8)로 컨쥬게이션하여 형광검출용 입자[QDs-anti hSAA1 (14F8)]로 사용하였다. 제작된 LFIA 스트립 위에 순차적으로 다른 농도의 hSAA1과 QDs-anti hSAA1 (14F8)의 복합체를 흘려주면, 테스트라인에 anti hSAA1 (10G1)/hSAA1/QDs-anti hSAA1 (14F8) 샌드위치 복합체가 형성되어 양자점에 의한 발광신호가 검출됨을 측정하였다. 최적화된 측방흐름이 가능한 완충용액 조건에서 100 nM 농도의 hSAA1 단백질의 유무를 5 min 안에 눈으로 확인 가능하였다.

• IMMUNE NETWORK
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• 제10권6호
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• pp.239-246
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• 2010

Background: Monoclonal antibodies (mAbs) recognizing Class III epitope of CD34 are essential for flow cytometric diagnosis of leukemia. Methods: 27H2 mAb was developed from a mouse alternatively immunized with human acute leukemia cell lines, KG1 and Molm-1. Using flow cytometric analysis of various leukemic cell lines and peripheral blood, immunohistochemical study of frozen tonsil, we characterized 27H2 mAb. Antigen immunoprecipitated with 27H2 mAb immunobloted with anti-CD34 mAb. A case of bone marrow sample of acute lymphoblastic leukemia (ALL) patient was obtained at CBNU Hospital. For epitope identification enzyme treatment with neuraminidase and O-sialoglycoprotein endopeptidase (OSGE) and blocking assay with known classIII mAb (HPCA-2) were done. Results: Only KG1 and Molm-1 revealed positive immunoreactivity. Immunohistochemical staining disclosed strong membranous immunoreactivity on high endothelial venules. Antigen immunoprecipitated by 27H2 mAb showed approximately 100 kDa sized band immunoblotted with anti-CD34 under non-reducing conditions. Epitope recognized by 27H2 mAb disclosed resistancy to both neuraminidase and OSGE treatment and completely blocked with known class III mAb preincubation. CD34 positive leukemic cells in BM of pre B cell ALL patient detected by FITC-conjugated 27H2 and HPCA-2 were identified with similar sensitivity. Conclusion: A novel murine mAb recognizing class III epitope of human CD34 with high affinity, which is useful for flow cytometric diagnosis of leukemia, was developed.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2008년도 Proceedings of the Convention
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• pp.77-86
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• 2008

It's been known that overexpression of the oncoprotein Her2 (eu/ErbB2), transmembrane receptor protein, occurs in human breast cancer. Her2-positive breast cancer patients who have Her2 overexpression show less therapeutic efficacy with enhanced metathesis and increased resistance to chemotherapy. So far, a humanized monoclonal antibody against Her2 protein called Herceptin is the only drug approved by Food and Drug Administration for treatment of Her2-overexpressing breast tumors. However, antibody therapy of Herceptin may not be ideal method for therapeutic intervention of Her2 protein expression. The therapeutic intervention of Her2 protein expression may be more efficiently achieved by inhibiting the expression of Her2 gene rather than by down-regulating the Her2 protein already overexpressed. Here, we found that the interaction of two proteins of ESX (an epithelial-restricted transcription factor) and DRIP130/CRSP130/Sur2 (a Ras-linked subunit of human mediator complexes) mediates the expression of Her2 gene. The association of ESX with Sur2 is mediated by a small hydrophobic face of 8-amino acid helix in ESX, suggesting that the ESX-Sur2 interaction can be a new novel target for Her2-positive cancer. The process to develop potent ESX-Sur2 interaction inhibitors targeting for Her2-positive cancer therapeutics will be discussed.

• Journal of Microbiology and Biotechnology
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• 제18권9호
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• pp.1606-1612
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• 2008

Interleukin (IL)-32 is a recently identified proinflammatory cytokine that is one of the IL-18 inducible genes, and plays an important role in autoimmune and inflammatory diseases. We produced antibodies against IL-32 and studied the expression of IL-32 in human stomach cancer. We detected IL-32 secreted from K-562 cells which were stably transfected with IL-32 and in the sera of stomach cancer patients by a sandwich ELISA using a monoclonal antibody KU32-52 and a polyclonal antibody. In order to optimize a sandwich immunoassay, recombinant IL-32a was added, followed by the addition of a biotinylated KU32-52 into microtiter plate wells precoated with a goat anti-IL-32 antibody. The bound biotinylated KU32-52 was probed with a streptavidin conjugated to HRP. This sandwich ELISA was highly specific and had a minimal detection limit of 80 pg/ml (mean${\pm}$SD of zero calibrator) and measuring up to 3,000 pg/ml. This ELISA showed no cross-reaction with other cytokines such as hIL-1$\alpha$, hIL-1$\beta$, hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-$\alpha$. Intra-assay coefficients of variation were 18.5% to 4.6% (n=10), and inter-assay coefficients were 23% to 9% (n=10). The average IL-32 level in the sera of 16 stomach cancer patients (189 pg/ml) was higher than that of 12 healthy control men (109 pg/ml). Our results indicate that serum IL-32 level can be detected by using an established ELISA, and that this immunoassay and mAb KU32-09 specific for immunohistochemistry can be used in the detection of expressed and secreted IL-32 in stomach cancer patients.

• 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.

• KSBB Journal
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• 제10권2호
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• pp.204-211
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• 1995

인지질 그 자체만으로는 안정한 이중층 리포좀을 형성하지 못하는 불포화 PE(DOPE)에 palmitoyl가 가 결합된 항체(p-IgG)를 지질층에 삽입시켜 lmm t unoliposome을 제조하고 그 특성에 관하여 살펴 보 았다. 우선 안정된 리포좀을 제조하기 위해서 고려 해야할 인자들로 항체 가공방법, 지질과 항체와의 몰 비, 그리고 각종 첨가제들에 대한 최적 조건을 조 사하였다. 예를 들면 p-IgG와 lipid의 볼비를 $2.5{\times}10^{-4}$ 으로 했을 때 안정한 리포좀을 만들 수 있었으며, 첨가제로 들어가는 DOC의 경우 최종 농도가 O.09wt % 일 때 calcein의 포집률이 최대가 되었고 c calcein의 최종 pH는 8.5~9.5 정도에서 안정한 라포좀이 제조될 수 있었다. 다중 빛 단일클론의 항체 를 삽입한 리포좀을 표면항원을 가진 표적세포와 결합시켰을 때 리포좀이 와해되면셔 포집된 calcein이 방출되는 것으로 보아 삽입된 p-IgG가 PE 리포좀을 형성하는데 필수척임을 알 수 있었다. 또 같은 리포좀을 비특이적인 세포와 접촉시켰을 때에는 아무 런 변화를 보이지 않아 calcein 방출이 항원-항체 반응에 의한 것임을 알 수 있었고 이로부터 표적 민 감성 PE 리포좀이 만들어졌음을 확인하였다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.94-94
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• 1997

The objective of this study was to generate and characterize monoclonal antibodies against rat airway mucins, and therefore, should serve as a useful tool in studying the regulation of airway mucins using various in vivo rat models that are currently available. As an antigen, we used a high molecular mass mucin preparation purified from the spent media of rat tracheal surface epithelial cells in primary culture. Seven hybridomas were obtained which secrete monoclonal antibodies against the rat mucin among which mAbRT03 showed the highest immunoreactivity against the mucin based on ELISA. All of the antibodies secreted by these hybridomas recognized carbohydrate epitopes but not sialic acid residues since their immunoreactivity was completely abolished by treatment of the mucin with 20 mM periodate but not with neuraminidase. Further characterization of mAbRT03 showed that: (1) it belongs to the IgM type, (2) it binds to high molecular mass mucins based on both Western blot analysis and indirect immunoprecipitation, (3) it binds to the luminal side of tracheal epithelium as well as some goblet cells based on immunohistochemistry, and (4) it also recognizes in vive airway mucins from rats but not from human nor hamsters which have been purified from the airway lavage fluids. This is the first anti-rat airway mucin monoclonal antibody which has been developed against purified rat airway mucins. Therefore, mAbRT03 should be able to serve as an invaluable tool in studying the regulation of airway mucins using various intact rat models that are currently available.

• 환경위생공학
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• 제4권1호
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• pp.17-28
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• 1989

Having studied the production of monoclonal antibodies for developing a diagnosis medicine which shall be detected by a high-sensitivity test by using Rhodotorula rubra as a fungi-host which had been extracted through biochemical tests and follow-up examinations on Yeast-like fungi obtained from pulmonary tissues of pulmonary tuberculosis patients who had been in Kong ju National Tuberculosis Hospital from Jun. to Dec. in 1987, I. have gained such results as follows: 1. The fusion rate was influenced by feeder cell layers, cell density and time required to the cell fusion with cells in myelona subculture. 2. The fusion rate did not show any significant difference when the cell was applyed with two molecular weights, i.e., 1500 and 4000, of polyethylene glycol. 3. Fused cells after the addition of HAT selection media were bright and round, whereas unfused myelona cells and spleen cells were shrunk and granulated. 4. The cell fusion rate turned out to be about $57.2\%$(150 wells / 264 wells). 5. $10\%$(15 wells / 150 wells) of the positive reaction was detected in monoclonal antibody screening. 6. The titer which had reacted positively to Rhodotorula rubra fungal-host was 800 times in density after the gradual dilution of the produced monoclonal antibodies with Indirect ELISA method. 7. The Strongest specific reaction came out after the peroxidase labelled anti-human Immunogobulin had been applyed to Rhodotorula rubra for activating its nature after making drift with Carbonate-bicarbonate buffer (pH 9.6) and drying completely.

• Radiation Oncology Journal
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• 제34권1호
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• pp.1-9
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• 2016

Overexpression of human epidermal growth factor receptor 2 (HER2) is found in about 20% of breast cancer patients. With treatment using trastuzumab, an anti-HER2 monoclonal antibody, systemic control is improved. Nonetheless, the incidence of brain metastasis does not be improved, rather seems to be increased in HER2-positive breast cancer. The mainstay treatment for brain metastases is radiotherapy. According to the number of metastatic lesions and performance status of patients, radiosurgery or whole brain radiotherapy can be performed. The concurrent use of a radiosensitizer further improves intracranial control. Due to its large molecular weight, trastuzumab has a limited ability to cross the blood-brain barrier. However, small tyrosine kinase inhibitors such as lapatinib, has been noted to be a promising agent that can be used as a radiosensitizer to affect HER2-positive breast cancer. This review will outline general management of brain metastases and will focus on preclinical findings regarding the radiosensitizing effect of small molecule HER2 targeting agents.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2001년도 추계학술대회 및 정기총회
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• pp.102-102
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• 2001

Taurine (2-ethaneaminosulfonic acid) is one of the major intracellular ${\beta}$ -amino acids in mammals and is required for a number of biological processes including membrane stabilization, osmoregulation, antioxidation, detoxification, modulation of calcium flux and neurornodulation. The taurine transporter (TAUT) which contains 12 hydrophobic membrane-spanning domains has been cloned from dog kidney, rat brain, mouse brain, human thyroid, placenta and retina. In this study, The TAUT cDNA from the human intestinal epithelial cell, HT-29 was cloned and sequenced. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to amplify partial cDNA encoding human intestinal TAUT. The coding region of the PCR product was 732 bp long. The primers were designed to encode highly conserved amino acid sequences near the transmembrane domains III (IPYFIFLF) and Ⅵ (KYKYNSYR) both in human and mouse. The TAUT cDNA amplified was ligated into the pGEX 4T-1 expression vector. The resulting sequence of human intestinal TAUT cDNA (Accession number of NCBI Genebank is AF346763) was identical to the sequences of the TAUTs previously determined in the human placenta and retina except 3 base pairs from that of the reported human thyroid. TAUT specific antibodies were generated to use them as biological tools in the studies of the biological role of TAUT. Peptides of 149-162 amino acid residue (14 amino acids) of the TAUT were synthesized. The synthetic peptide used in this study was LFQSFQKELPWAHC. This region was chosen not only to avoid putative glycosylation sites but also to exclude regions of known homology with GABA transporters in the extracellular hydrophilic domains. The synthetic peptide, TAUT-1 was conjugated with carrier protein, kehole lympet hemocyanin (KLH) to use as an antigen. When used for immunization on a rabbit to produce polyclonal antiserum, the conjugates elicited high -titered specific anti-TAUT-1 antibodies, which reacted well with the ovalbumin (OVA) conjugated peptides in ELISA. The KLH-conjugated peptide was also used as immunizing antigen in BALB/c mice to produce TAUT specific monoclonal antibodies. From the culture supernatant of the hybridoma, the specificity of anti-TAUT-1 monoclonal antibodies was confirmed by ELISA. Further applications of more tools in TAUT expression analysis will be performed such as western blotting and flow cytometry.