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

Michung Yoon(윤미정) 대한의생명과학회 1997 Biomedical Science Letters 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. 친화크로마토그래피법과 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 (EUSA) 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.

Measurement of the Affinity Constant of Monoclonal Antibody to Human Apolipoprotein A-I by ELISA

Michung Yoon(윤미정),Hyun-Hee Lee(이현희) 대한의생명과학회 1995 Biomedical Science Letters Vol.1 No.1

본 연구에서는 효소면역진단법을 이용하여 사람의 아포지단백질 A-I에 대한 단일클론항체의 해리상수(Kd)를 측정하고자 하였다. 먼저 단일클론항체와 항원을 평형에 도달할 때까지 액체상에서 반응시킨 후, 평형상태에서 항원과 결합하지 못하고 남아있는 항체를 미세적정판에 결합되어 있는 항원과 반응시킨다. 그 다음 결합된 항체의 양이 효소면역분석법에 의하여 측정한다. 본 실험방법을 이용하여 측정된 아포지단백질 A-I에 대한 단일클론항체의 해리상수는 정제된 형태의 경우 0.625×10??이었으며, 정제되지 않은 하이브리도마 배양액의 경우 0.720×10??이었다. 이 방법은 간단하고 재생성이 높으며, 정확하고 방사선 동위원소를 사용하지 않는 등 많은 장점을 가진 것으로 생각된다. The present study was undertaken to determine the dissociation constant (Kd) of monoclonal antibody to human apolipoprotein A-I (apo A-I) using enzyme-linked immunosorbent assay (ELISA). First the monoclonal antibody was incubated in solution with the antigen until the equilibrium was reached; then the free antibody which remains unsaturated at equilibrium was captured by binding to antigen on the micro titer plate and be measured by a classical indirect ELISA. The value of Kd determined from Scatchard plot was 0.625×10?? for purified antibody and 0.720×10?? for unpurified antibody. This method was valuable for the measurement of true dissociation constant and found to be simple, reproducible, and accurate.

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.

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.

『東醫寶鑑』의 熟地黃 제조방법

노종성,윤미정,신순식,Roh, Jong Seong,Yoon, Michung,Shin, Soon Shik 대한한의학방제학회 2016 大韓韓醫學方劑學會誌 Vol.24 No.1

Objectives Rehmanniae Radix Preparata was prepared in the traditional Rehmanniae Radix Preparata preparation method set forth in the 『Treasured Mirror of Eastern Medicine(Donguibogam)』 with a view to measuring the contents of 5-Hydroxymethyl-2-furaldehyde(5-HMF) at individual stages of steaming and sundrying and identifying new chemical components.Methods Based on the traditional Rehmanniae Radix Preparata preparation method set forth in the 『Treasured Mirror of Eastern Medicine』, Rehmanniae Radix Preparata steamed and sundried once through nine times was prepared. Thereafter, 5-HMF contents were analyzed and new chemical components were identified in the Rehmanniae Radix Preparata using Waters HPLC e2695, 2640 detectors, a Waters Acquity UPLC system, and a Micromass Q-TOF Premier mass spectrometer.Results The Rehmanniae Radix Preparata preparation method set forth in the 『Treasured Mirror of Eastern Medicine』 is a unique preparation method in Republic of Korea different from that in China. In the first stage of the method, fresh Rehmanniae Radix Crudus was divided into high quality, medium quality, and low quality ones named Rehmanniae Radix Crudus (Caelum)(天黃), Rehmanniae Radix Crudus (Homo)(人黃), and Rehmanniae Radix Crudus (Terra)(地黃) respectively to use Rehmanniae Radix Crudus (Caelum) and Rehmanniae Radix Crudus (Homo) for preparation of juice while using Rehmanniae Radix Crudus (Terra) to make Rehmanniae Radix Preparata. In the second stage, Rehmanniae Radix Crudus (Caelum) and Rehmanniae Radix Crudus (Terra) were made into juice and Rehmanniae Radix Crudus (Terra) was soaked in the juice. In the third stage, among auxiliary materials, rice wine named Purum Vinum Oryzae(淸酒) brewed from sticky rice was sprinkled on Rehmanniae Radix Crudus (Terra) to the extent that Rehmanniae Radix Crudus (Terra) became wet. In the fourth stage, Rehmanniae Radix Preparata steamed in earthenware steamer was dried under natural sunlight. The contents of 5-HMF in Rehmanniae Radix Preparata steamed and sundried once through nine times were shown to be below 0.1% in all cases. Pomolic acid was identified as a new chemical component.Conclusions In conclusion, the Rehmanniae Radix Preparata preparation method set forth in the 『Treasured Mirror of Eastern Medicine』 is thought to be a unique preparation method in South Korea in which Rehmanniae Radix Preparata is completed through the first stage in which fresh Rehmanniae Radix Crudus collected from fields is divided into high, medium, and low quality ones and fresh Rehmanniae Radix Crudus juice is made, the second stage in which the high quality fresh Rehmanniae Radix Crudus is soaked in the fresh Rehmanniae Radix Crudus juice, the third stage in which the fresh Rehmanniae Radix Crudus is steamed, and the fourth stage in which the steamed Rehmanniae Radix Crudus is dried.

대황목란탕(大黃牧丹湯)의 배오(配伍)분석과 그 약리활성

김도회,윤미정,신순식,Kim, Do Hoy,Yoon, Michung,Shin, Soon Shik 대한한의학방제학회 2018 大韓韓醫學方劑學會誌 Vol.26 No.1

Objectives : I analysed daehwangmokdan-tang's compatibility principle by the system of chief, deputy, assistant, and envoy and investigated pharmacological activities by categorizing with chemical components, molecular level, cellular level, animal level and human level based on Korean and Chinese studies for this formula. Methods : Daehwangmokdan-tang's compatibiltity principle was examined by the system of chief, deputy, assistant, and envoy. I looked into studies that presented in Korea from 1956 to 2016 about daehwangmokdan-tang through KOREA INSTITUTE OF ORIENTAL MEDICINE, Korean medicine information system (OASIS) and in Chinese for 20 years about daehwangmokdan-tang through China National Knowledge Infrastructure, CNKI. Then classify into chemical components, molecular level, cellular level, animal level and human level to analyse. Results : According to the system of chief, deputy, assistant, and envoy, chief herb is Rhei Radix et Rhizoma and Moutan Cortex, deputy herb is Natrii Sulfas and Persicae Semen, assistant and envoy herbs are Trichosanthis Semen. The amount of extraction of paeonol, total anthraquinone, and conjugated anthraquinone from daehwangmokdan-tang with the formulation of the system of chief, deputy, assistant, and envoy was the highest, and in the formulation of chief herb and deputy herb, the extraction amount of paeonol and conjugated anthraquinone was the lowest. With other formulations, the amount of extraction of total anthraquinone and conjugated anthraquinone was improved, although the degree was different. In particular, when it is blended with Persicae Semen as a deputy herb, the extraction amount of total anthraquinone and conjugated anthraquinone of Rhei Radix et Rhizoma as a chief herb is greatly increased, and the extraction amount of paeonol is rather different, but it is lowered. It was found that the amount of Mg Ca K Na in daehwangmokdan-tang was the highest. Pharmacological activities can be detected in inflammatory mediators and enzymes for molecular level. For cellular level, it can be determined in lipopolysaccharide (LPS)-stimulated RAW 264.7 cell line. In mouse and rats for animal level and human level, in inflammatory diseases (acute appendicitis, acute pancreatitis, acute cholecystitis, acute abdominal disease, ect.), pharmacological activities was caught. Conclusions : From the above results, daehwangmokdan-tang is composed in line with the system of chief, deputy, assistant, and envoy, suggesting that there is certain rationality and scientific. Pharmacological activities of daehwangmokdan-tang are effective to anti-inflammation, improvement of sepsis, analgesic, muscle relaxation, and improvement of intestinal flora and its metabolites. Daehwangmokdan-tang is consistent with the action of diuresis and anti-inflammation in terms of the content of elements, suggesting that there is action of purging fire and removing blood stasis, defecation detoxification, cooling blood and clearing heat and activating blood and dispersing stasis.

계지복령환(桂枝茯苓丸)의 배오(配伍)분석과 그 생리활성

김도회,정양삼,윤미정,윤유식,신순식,Kim, Do Hoy,Jung, Yang Sam,Yoon, Michung,Yoon, Yoosik,Shin, Soon Shik 대한한의학방제학회 2016 大韓韓醫學方劑學會誌 Vol.24 No.4

Objectives : We analysed Gyejibongnyeong-hwan's compatibility principle and investigated biological activities by categorizing with molecular level, cellular level, animal level and human level based on Korean study for this formula. Methods : Gyejibongnyeong-hwan's compatibiltity principle was examined by the system of chief, deputy, assistant, and envoy. We looked into studies that presented in Korea from 1956 to 2016 about Gyejibongnyeong-hwan through Korea Institute of Oriental Medicine, Korean medicine information system (OASIS). Then classify into molecular level, cellular level, animal level and human level to analyse. Results : According to the system of chief, deputy, assistant, and envoy, chief herb is Cinnamomi Ramulus, deputy herb is Persicae Semen, assistant herb is Moutan Cortex, Paeoniae Radix, Poria, and envoy herb is Mel. Biological activities can be detected in transcription factors, enzymes, and inflammatory mediators for molecular level. For cellular level, it can be determined in human uterine endometrial cancer cell, human hepatocarcinoma cell, and human platelets. In mouse and rats for animal level, in overian cystoma, menorrhalgia, quality of life improvement in postmenopausal women, and blood stasis with motor vehicle accident for human level, biological activities was caught. Conclusions : From above results, Gyejibongnyeong-hwan is composed in line with the system of chief, deputy, assistan, and envoy. Biological activities are effective to improvement of menorrhalgia, anti-cancer, anti-oxidative, anti-inflammation, improvement of atherosclerosis, analgesic, anti-convulsion, wound healing, and improvement of liver function.

輕身解脂丸 (GGT1)이 형질전환 비만모델 hGHTg 수컷 쥐의 비만관련 유전자 발현에 미치는 영향

정양삼(Yang Sam Jung),윤미정(Michung Yoon),김경철(Gyeong Cheol Kim),신순식(Soon Shik Shin) 한의병리학회 2006 동의생리병리학회지 Vol.20 No.1

암컷 hGHTg 비만 쥐에서 輕身解脂丸 (GGT1)에 의한 비만관련 유전자 mRNA 발현의 변화

윤기현(Ki Hyeon Yoon),윤미정(Michung Yoon),김훈(Hoon Kim),신순식(Soon Shik Shin) 한의병리학회 2006 동의생리병리학회지 Vol.20 No.2

고추의 나출 소포자 배양시 전처리 기간, 2-Hydroxynicotinic Acid 및 약-공동전처리가 소포자배 발생에 미치는 영향

박은준,김진애,이종숙,장인창,윤미정,정상호,김문자,Park Eun-Joon,Kim Jin-Ae,Lee Jong-Suk,Jang In-Chang,Yoon Michung,Chung Sang-Ho,Kim Moonza 한국식물생명공학회 2005 식물생명공학회지 Vol.32 No.1

고추의 나출 소포자로부터 배를 유기하는데 최적인 조건을 확립하기 위하여 micro-blender를 사용하여 소포자들을 나출한 후 NLN 배지에 배양하였으며 $32^{\circ}C$의 고온처리 기간, 전처리 배지내 2-hydroxynicotinic acid의 첨가, 그리고 약과 소포자의 공동전처리가 소포자배의 발생에 미치는 영향을 조사하였다. 나출 소포자들을 기아배지에서 고온처리 한 후 NLN 배지에 배양하였을 때 다수의 배가 발생하였다. 배양 3주 후부터 구형 및 어뢰형배가 관찰되었는데 이때 배의 발생은 비동조적으로 일어났다. 배양4주가 되면 구형과 어뢰형배 이외에 자엽배들이 발생하였다. 발생한 배들 중자엽배늘은 $2\%$ sucrose가 첨가되고 생장조절물질은 첨가되지 않은 B5 고체배지로 옮겼을 때 정상인 유식물들로 발달하였다. 고온처리 기간이 $1\~2$일로 짧은 경우 소포자배의 발생은 높았으나 대부분이 발달초기의 구형 또는 심장형배이었으며 자엽배의 발생은 매우 드물었다. 고온처리 3일 이상에서는 배의 발생은 크게 감소하였으나 자엽배가 다수 발생하였다. Inducer chemical로 알려진 2-hydroxynicotinic acid를 약전처리배지에 첨가하였을 때 배의 발생은 다소 높았으나 발달은 오히려 억제되어 대부분이 구형 또는 심장형이었다. 소포자 전처리시 약을 첨가한 경우 배의 발생이나 발달 모두 억제되었다. 본 연구결과 고추의 나출 소포자로부터 다수의 배를 획득하였고 식물체를 재분화 시키는데 처음으로 성공하였다. 이와 같은 소포자 배양시스템은 앞으로 더 많은 배를 생산할 수 있는 배양조건이 확립되어야 하지만 homozygous한 배가 반수체의 생산 뿐만아니라 형질 전환과 열성 또는 우성의 돌연변이체 선발에 매우 유용하게 이용될 수 있을 것이다. 비해 $ 0.27\~0.79\;\cal{mg/g}$ F.W.가 높았으며 환기횟수 $0.1 h^{-1}$ 처리구는 광도의 증가에 따른 효과가 나타나지 않았으나 환기횟수 $0.1 h^{-1}$ 처리구는 높은 광도에서 감소하였다. 환기횟수 $4.9 h^{-1}$는 $0.1 h^{-1}$에 비해 잎의 공변세포가 크고 주변 부세포가 잘 발달되어 있었다. 특히 PPF $99\;{\mu}mol\;m^{-2}s^{-1}$에서 환기횟수 $0.1 h^{-1}$는 부세포의 발달이 미흡하고 기공이 많이 열려 있는 상태인 반면 환기횟수 $4.9 h^{-1}$는 부세포가 잘 발달된 잎을 지니고 있었다.:PR30, KB50:PR50, PR100:KB0 처리구의 $1m^2$ 당 개체 수는 각각 16,600개, 6,700개, 4,900개, 3,300개, 12,400개였으며 총직립경 수는 각각 33,200개, 22,800개, 18,000개, 15,000개, 62,000개였다. 개체 수는 KB100:PR0처리구가 가장 높았고, 다음은 PR100:KB0처리구였으며 혼합처리구의 경우는 Kentucky bluegrass 혼합비 율이 높을수록 높았다. 총직립경 수는 PR100:KB0 처리구가 KB100:PR0 처리구보다 오히려 높았으며 혼합 처리구의 경우는 개체수와 비슷한 경향을 보였다. 혼합처리구의 경우 초종별 개체수의 비율은 KB80:PR20는 87:13, KB70:PR30는 78:22, KB50:PR50은 48:52의 비율로 나타났다. 조성시기의 기상과 피복율과의 상관관계 2001년 가을과 2002년 봄의 일일평균기온을 비교하여 보면 가을(9월, 10월, 11월) 3개월간의 일일평균기온은 Microspores were isolated from pepper (Capsicum annuum L.) anthers by using a micro-blender and cultured in modified NLN medium at $25^{\circ}C$. The influence of pretreatment period at $32^{\circ}C$, adding the 2-hydroxynicotinic acid to a pretreatment medium, and co-pretreatment anthers with microscopes on the induction of embryo were examined. Globular and torpedo embryos were observed from 3 weeks after culture. Embryo development was not synchronized within culture. After 4 weeks in culture, in addition to globular and torpedo embryos, cotyledonary embryos were observed. Normal cotylodonary embryos developed into plantlets when transferred to a solid hormone free B5 medium containing $2\%$ sucrose. Embryo yields were significantly higher after 1- and 2-day pretreatment at $32^{\circ}C$. However the development of embryo ceased at the globular or heart stage. In contrast, embryo yields were lower after 3- to 6-day pretreatment at $32^{\circ}C$ and embryo developed at the cotyledonary stage. After adding the 2-hydroxynicotinic acid to anther pretreatment solution, embryo yields were slightly increased. However most embryos occurred were at the globular or heart stage. Co-pretreatment of microspores with anthers was deleterious for embryo induction and development. AS far as we know, this is the first report of success in obtaining high frequency of embryogenesis and plantlets formation from isolated microspores of pepper. Although the culture conditions have to be optimized further, this promising microspore culture system can be used for genetic transformation, selection for dominant and recessive traits as well as for the production of homozygous doubled haploid plants.