Immunosuppression of peripheral lymphocytes and thymocytes during the implantation period in rats

Yoon, Michung 목원대학교 자연과학연구소 1994 自然科學 硏究論文集 Vol.3 No.1

본 연구에서는 모체가 수정란을 이물질로서 인지하는 최초의 시기인 착상시기에 모체의 면역반응을 알아보고, 착상시기에 증가하는 호르몬을 중심으로 면역반응을 변화시키는 인자를 조사하고자 하였다. 흰쥐의 착상시기인 임신 제 5일의 lymphocyte의 proliferation은 임신하지 않은 경우와 비교하여 크게 억제되었으며, 임신한 흰쥐의 혈청은 lymphocyte와 thymocyte 둘다의 proliferation을 억제하였다. 따라서 착상시기에 증가되는 혈청내 호르몬들의 영향이 조사되었을 때, estradiol, progesterone 및 prostaglandin F_2α 는 lymphocyte와 thymocyte의 proliferation에 영향을 주지 않았으나 prostaglandin E는 농도에 비례하여 억제하였다. 또한 prostaglandin합성의 억제제인 indomethacin을 처리하였을 때 억제되었던 lymphocyte와 thymocyte의 proliferation이 회복되는 것을 관찰하였다. 이러한 결과로 보아, 흰쥐의 착상시기에 면역반응이 억제되며, prostaglandin E가 면역반응을 조절하는 인자중의 하나로 생각된다. The present study was attempted to investigate maternal immune response during the implantation period when mother first recognizes the blastocyst as foreign body and determine the effects of hormones, which are known to be involved in the implantation process in rats, on the maternal immune response. Peripheral lymphocyte activities of pregnant rats on days 5, 6 and 8, implantation period and just after were stppressed to compare with those of non-pregnant rats. When sera of non-pregnant and pregnant rats on days 5 and 6 were added to cultures of non-pregnant rat thymocytes, pregnant rat serum inhibited thymocyte activities much more than non­pregnant rat serum. Progesterone inhibited peripheral lymphocyte activities at concentrations 10^-6 - 10^-5 M, pharmacological doses and estradiol even at more than a thousand times of physiological concentrations inhibited slightly peripheral lymphocyte activities. But prostaglandin E(PGE), not prostaglandin F_2α (PGF_2α ) significantly inhibited peripheral lymphocyte activities even with concentrations of 3×8exp(-8)-3×10exp(-6)M, physiological doses and when PGE was added to cultures of non-pregnant rat thymocytes, thymocyte activities were significantly inhibited. The treatment of indomethacin (ID) at doses of 0.1 or 1.0㎍/ml tended to enhance lymphocyte activities which was depressed on day 8 of pregnancy, 28 or 23%, respectively. Although in non-pregnant rats, enhancement of lymphocyte response was shown after the treatment of ID, this enhancement was much less than that in pregnant rats. It is, therefore, concluded that maternal immune response was suppressed during the implantation process and PGE might be one of factors which modulate immune response in rats.

Production of a Monoclonal Antibody to Human a-Fetoprotein and Development of Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assays for Human α-Fetoprotein

Michung Yoon(윤미정),Hyun-Hee Lee(이현희),Youngwon Lee(이영원) 대한의생명과학회 1999 Biomedical Science Letters Vol.5 No.1

본 연구에서는 혈장이나 양수에 있는 α-fetoprotein (AFP)을 인식할 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 κ 경사슬의 isotype을 나타냈다. 또한 immunoblotting 방법과 ELISA로 특이도를 조사한 결과 모노클로날 항체는 AFP와만 반응하였고, 결합 친화상수는 0,8×10?¹? 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. This study was attempted to generate a monoclonal antibody against human α-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/O-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 κ 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 0.8×10?¹? 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 immunodiagnostic kit for the measurement of AFP concentration.

Production of Polyclonal Antibody against α-Fetoprotein and Polyclonal Antibody-Based Competitive Enzyme-Linked Immunosorbent Assay for α-Fetoprotein

Yoon,Michung THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1997 Journal of biomedical laboratory sciences Vol.3 No.2

인간 α-fetoprotein (AFP)은 간암, 위암, 생식기종양 및 신경관이상인 환자를 검사하고 진단하는데 유용한 지표로 알려져 있다. 본 연구에서는 사람의 AFP를 분리정제하여 폴리클로날 항체를 생산하고 인간 혈장과 양수내의 AFP를 측정하기 위한 경쟁적 효소면역분석법을 개발하고자 하였다. 친화 크로마토그래피법과 SDS-polyacrylamide 전기영동법을 이용하여 양수로부터 AFP를 분리하였다. 정제된 AFP를 토끼에 주사하여 폴리클로날 항체를 생산하였으며, 이중면역확산법과 Western blot 분석법을 사용하여 본 연구실에서 제조된 항체의 항원 특이성이 대단히 높음을 확인하였다. AFP와 항혈청을 이용하여 표준곡선을 얻었으며, 민감도는 5ng/ml이었고, 작용범위는 5∼1,000ng/ml이었다. 분석내 CV는 4.5%이었고, 분석간 CV는 8.5%이었다. 따라서 이러한 결과로 보아 본 연구에서 개발된 경쟁적 효소면역분석법이 AFP를 측정하기에 적절하며, 간암 등의 기초연구에도 많은 기여를 할 것으로 생각된다. α-Fetoprotein (AFP) has been a useful marker in screening and/or monitoring patients with hepatocellular carcinoma, gonadal germ cell tumor, gastric carcinoma and neural tube defects. In the present study, it was attempted to produce anti-human AFP polyclonal antibodies and to develop a competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of AFP in human plasma and amniotic fluid. AFP was isolated from amniotic fluid using an isolation procedure consisting of affinity chromatography and preparative polyacrylamide gel electrophoresis. The antibody directed against AFP was raised in rabbits. Double immunodiffusion and Western blotting methods showed that the antiserum was highly specific, reacting with only AFP-containing samples. Standard curve was obtained by using purified AFP and specific antiserum. The assay sensitivity was 5ng/ml and the working range was 5∼1,000ng/ml. The within-assay and between-assay coefficient of variance (CV) was 4.5% and 8.5%, respectively. These results indicate that the assay is valuable for the measurement of AFP and found to be simple, reproducible, and accurate.

Production of a Monoclonal Antibody to Human α-Fetoprotein and Development of Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assays for Human α-Fetoprotein

Yoon,Michung,Lee,Hyung-Hee,Lee,Youngwon THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1999 Journal of biomedical laboratory sciences Vol.5 No.1

본 연구에서는 혈장이나 양수에 있는 α-fetoprotein (AFP)을 인식할 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 ×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. This study was attempted to generate a monoclonal antibody against human α-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/O-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 ×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 immuno-diagnostic kit for the measurement of AFP concentration.

사람의 혈중 아포지단백질 A-1(apo A-1) 측정을 위한 경쟁적 효소면역 분석법 (ELISA) 의 개발

Michung Yoon,Ju Won Kwak,Moon Hi Han 한국지질동맥경화학회 1993 韓國脂質學會誌 Vol.3 No.1

Synthetic Peptide-Based Enzyme-Linked Immunosorbent Assay for Human α-Fetoprotein

Yoon, Michung,Lee, Hyun-Hee 대한의생명과학회 2001 Biomedical Science Letters Vol.7 No.3

α-Fetoprotein (AFP) is a good marker for the detection of several diseases such as hepatocellular carcinoma, gonadal germ cell tumor, gastric tumor, and Down's syndrome. In this study, we developed ELISA, using synthetic peptides corresponding to the epitopes. of AFP. Five kinds of peptides were synthesized from AFP to produce antibodies in rats that recognize AFP in human plasma as well as amniotic fluid and do not cross-react with serum albumin. All five kinds of antibodies showed good reactivities with their peptide-keyhole limpet hemocyanin conjugates. Anti-synthetic peptide 1 (R-N-E-Y-G-I-A-S-I-L, 4-13) antibody, in particular, reacted well with AFP as well as synthetic peptide 1-KLH but not with human serum albumin. The binding affinity (Kd) was 2.7X10^-9 M for peptide 1 and 6.8X10^-8 M for AFP. The range for measurement of AFP was 10∼1,000 ng/ml. The within-assay and between-assay coefficients of variance (CV) were 4.83% and 10.97%, respectively. In a sample of 31 sera and 33 amniotic fluids, there was a good correlation between AFP values determined in this assay and those in a commercial kit. These results indicate that the antibodies against synthetic peptides corresponding to the epitopes of AFP are highly specific to AFP and synthetic peptide-based ELISA would be useful for the measurement of human AFP.

Synthetic Peptide-Based Polyclonal Antibody Production for Lipoprotein〔a〕

Yoon, Michung 목원대학교 자연과학연구소 1997 自然科學 硏究論文集 Vol.6 No.-

여러 종류의 apo〔a〕를 인지하고 plasminogen과의 교차반응이 없는 항체를 생산하기 위하여 apo〔a〕kringle 4의 공통부위에 해당하는 10-mer peptide(T-T-V-T-G-R-T-C-Q-W)를 합성했다. 전달 단백질인 keyhole limpet hemocyanin과 결합된 합성 peptide를 토끼에 주사하여 항혈청을 얻었으며, ELISA와 immunoblotting 분석결과 항체들은 Lp〔a〕, 사람의 혈청 및 전체 lipoprotein 분획물 모두와 좋은 반응을 보여 주었다. 그러나 plasminogen과도 반응성을 나타냈으므로 10-mer peptide가 결합된 column에 항혈청을 통과시켜 항체를 정제하였으며 정제된 항체는 Lp〔a〕와 잘 반응하였고 동시에 plasminogen과는 반응하지 않았다. 따라서 본 연구에서 생산된 항체는 혈장 Lp〔a〕측정을 위한 면역진단시약의 개발에 유용할 것으로 생각된다. In order to produce an antiserum that commonly recognizes different forms of apo〔a〕 with a minimum cross-reactibity to plasminogen, I synthesized a 10-mer peptide(T-T-V-T-G-R-T-C-Q-W) corresponding to the conserved region of apo〔a〕kringle 4. The peptide was conjugated to a carrier protein, keyhole limpet hemocyanin and the conjugate was used to raise antiserum fro rabbits. The antibodies thus obtained reacted well with native Lp〔a〕fraction (1.05<d<1.08) of human serum as well as with total lipoprotein-enriched fraction (d<1.21), when analyzed by ELISA and immunoblotting. Unexpectedly, the whole antiserum showed a considerable cross-reaction with plasminogen in the ELISA while no cross-reaction was detectable in immunoblotting. The antiserum was affinity-purified through the 10-mer peptide-conjugated Sepharose column and the resulting IgG fraction was found out to be very specific to Lp〔a〕, showing no detectable cross-reaction with plasminogen, in ELISA analysis. The antiserum in this study would be useful for future development of an immunodiagnostic kit for measurement of plasma Lp〔a〕concentration.

Development of a Monoclonal Antibody to Human α-Fetoprotein : Characterization of a Monoclonal Antibody with High Specificity and High Binding Affinity 높은 특이도와 결합력을 가진 항체의 성격 규명

Yoon, Michung 목원대학교 자연과학연구소 1998 自然科學 硏究論文集 Vol.7 No.-

본 연구에서는 혈장이나 양수의 α-fetoprotein(AFP)을 인식할 수 있는 모노클로날 항체를 제조하고, 모노클로날 항체의 성격을 규명하고자 하였다. 양수로부터 얻은 AFP를 쥐에 주사한 후 비장세포를 분리하여 종양세포(Sp2/O-Ag-14)와 융합하였고, 하이브리도마 기술을 이용하여 모노클로날 항체를 제조하였다. 모노클로날 항체를 클로닝하였으며, 생성된 항체를 MabF22로 명명하였고, MabF22의 isotyping 분석은 IgG1 중사슬과 k 경사슬을 나타냈다. 또한 immunoblotting 방법과 ELISA로 특이도를 조사한 결과 모노클로날 항체는 AFP와만 반응하였고, 결합 친화상수는 0.8x10^-10 M이었다. (본 연구는 1997년도 목원대학교 교내연구비의 보조에 의한 것임.) This study was attempted to generate a monoclonal antibody against human α-fetoprotein(AFP), 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/O-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 0.8x10^-10 M.

The Role of Angiogenesis in Obesity

Michung Yoon(윤미정) 한국생명과학회 2014 생명과학회지 Vol.24 No.5

혈관신생은 모든 조직의 성장과 발달, 그리고 상처회복 등에 매우 중요하다. 지방조직은 우리 몸에서 가장 혈관이 발달된 조직으로서 각 지방세포들은 모세혈관에 둘러싸여 있으며 신생혈관들은 지방세포에 영양분과 산소를 공급한다. 혈관의 내피세포들은 파라크린 신호경로, 세포외 성분, 세포들 간의 직접적인 작용을 통해 지방세포와 교류한다. 활성화된 지방세포들은 VEGF, FGF-2, leptin, HGF와 같은 혈관신생인자들을 생성하며, 이들은 단독으로 혹은 협력하여 혈관신생을 증가시키고 지방조직의 성장과 대사를 촉진한다. 따라서 혈관신생 억제제들은 비만과 비만관련 질환을 치료하는데 유용할 것으로 생각된다. Angiogenesis, the formation of new capillary blood vessels, is a tightly regulated process. Under normal physiological conditions, angiogenesis only takes place during embryonic development, wound healing, and female menstruation. Dysregulation of angiogenesis is associated with many diseases, such as cancer, rheumatoid arthritis, psoriasis, and proliferative retinopathy. The growth and expansion of adipose tissue require the formation of new blood vessels. Adipose tissue is probably the most highly vascularized tissue in the body, as each adipocyte is surrounded by capillaries, and the angiogenic vessels supply nutrients and oxygen to adipocytes. Accumulating evidence shows that capillary endothelial cells communicate with adipocytes via paracrine signaling pathways, extracellular components, and direct cell-cell interactions. Activated adipocytes produce multiple angiogenic factors, including VEGF, FGF-2, leptin, and HGF, which either alone or cooperatively stimulate the expansion and metabolism of adipose tissue by increasing adipose tissue vasculature. Recently, it was demonstrated that antiangiogenic herbal Ob-X extracts and Korean red ginseng extracts reduce adipose tissue mass and suppress obesity by inhibiting angiogenesis in obese mice. Thus, angiogenesis inhibitors provide a promising therapeutic approach for controlling human obesity and related disorders.

Isolation of α-Fetoprotein and Production of Polyclonal Antibody against α-Fetoprotein

Yoon, Michung,Lee, Hyun-Hee 목원대학교 자연과학연구소 1996 自然科學 硏究論文集 Vol.5 No.-

Human α-fetoprotein (hAFP) has been isolated from amniotic fluid using an isolation procedure consisting of only two major steps: affinity chromatography followed by preparative polyacrylamide gel electrophoresis (PAGE). Isolation efficiency was 51% and the final product appeared homogenous on the basis of three criteria for purity. The antibody directed against hAFP antigen was raised in rabbits. The purity and specificity of the antiserum were checked by double immunodiffusion and Western blotting methods. In double immunodiffusion, the antiserum formed single immunoprecipitin line with each of the several hAFP containing samples. In Western blotting analysis, positive band was at a molecular weight-position of 66Kd when hAFP or amniotic fluid was loaded on the gel. These data indicate that hAFP and its antibody were well-prepared for the development of hAFP assay system.