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肝癌細胞의 γ-Glutamyltranspeptidase에 對한 Monoclonal antibody에 關한 硏究
金明坤,柳總根 고려대학교 의과대학 1992 고려대 의대 잡지 Vol.29 No.2
This study was devised to produce monoclonal antibody against hepatocelullar carcinoma speclfic γ-glutamyltranspeptidase (HCC GGT), which proceeded by first purifying the HCC GGT using ion exchange chromatography and immunoaffinity chromatography. Subsequently, a mouse was immunized with the purified enzyme, and then spleen cells taken from the immunized mouse was fused with mouse myeloma cells (SP2-Ag 14) to form the hybridoma. The following results were obtained by characterization of the monoclonal antibody of the hybridoma using immunoglobulin isotyping and immunoblot. 1. The purification of HCC GGT by means of ion exchange chromatography and immunoaffinity chromatogiaphy produced a yield of 29.41%, a purification fold of 167.87, and final specific activity of 21units per mg of protein. 2. Direct enzyme linked immunoadsorbent assay (ELISA) method was used to measure the antibody level against HCC GGT. An antibody titration of the blood sample taken from the mouse immunized with the above enzyme showed value over 1:5, 120. 3. The cell fusion of the mouse myeloma cell and the spleen cell of the immunized mouse was generated 14 positive wells in 221 wells. (specific fusion efficacy of 6.33%) Only one IgG2a monoclonal antibody was determined by both repetitive cloning procedures and immunoglobulin subisotyplng. 4. A antigen-antibody cross matching after the western blot revealed a specific reaction between the HCC GGT antigen at the 40Kd band and the monoclonal antibody of the hybridoma. And the monoclonal antibody did not react with GGT derived from normal liver or kidney.
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단일클론 항γ-Glutamyltransferase항체를 이용한 효소 및 방사 면역측정법 적용에 관한 연구
金明坤,柳總根 고려대학교 의과대학 1994 고려대 의대 잡지 Vol.31 No.1
γ-Glutamyltransferase (GGT: E. C. 2. 3. 2. 2.) is a glycoprotein enzyme which is involved in glutathione metabolism and amino acid transport through the plasma membrane. It is distributed widely in several organs including liver and kidney. Several isozymes of GGT have been reported and some of the isozymes may be associated with hepatocarcinogenesis. We have produced six monoclnal antibodies (mAbs) against GGT purified from the liver of 2-acetamidofluorene (AAF) treated rats. All of the six mAbs were obtained by immunizing mice with liver GGT. Liver GGT from AAF treated rats was biochemically purified to a specific activity of 125.6 units per mg of protein. The overall purification was 551 folds and the final yield was 20. 8. Six hybridomas which produced anti-GGT Abs were extensively subcloned and injected into the peritoneal cavity of BALB/c mice to obtain large quantities of Abs. These mAbs were purified from ascites by ammonium sulfate precipitation and protein A sepharose CL-4B column chromatography. Using these mAbs we performed enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC), and autoradiography (ARG) to study the distribution of GGT isozymes in tissue. The results indicate that GGT-mAb 1 is specific for the AAF treated liver GGT, GGT-mAb 5 for the normal liver GGT, and GGT-mAb 6 for the normal kidney GGT. These mAbs may be used to evaluate the distribution of GGT isozymes in different tissues.
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동면중 짱뚱어 근육유(筋肉油)의 지방질과 중성 및 인지질 조성의 변화
김명곤,백승화,최선남,김종배,박일웅,Kim, Myung-Kon,Baek, Seung-Hwa,Choe, Sun-Nam,Kim, Jong-Bae,Park, Il-Woong 한국응용생명화학회 1998 Applied Biological Chemistry (Appl Biol Chem) Vol.41 No.7
동면시 짱뚱어의 에너지 대사에 관한 정보를 얻고자 근육 중 지방질과 지방산조성을 성숙기(8월), 동면직전(11월), 동면직후(4월)로 나누어 분석 검토하였다. 조지방과 중성지질 조성은 각각 8월 1.2, 68.3%에서 11월 0.7, 53.8%, 4월 0.4, 42.6%로 점차 줄었고, 인지질과 당지질은 8월 29.5, 2.2%에서 4월 52.1%, 5.3%로 각각 증가 하였다. 중성지질 중 TG함량은 8월 53.8%에서 11월 33.6, 4월 23.1%로 점차 줄었으나, FFA와 sterol류는 반대로 8월 13.5, 14.2%에서 4월 22.3, 24.5%로 각각 증가하였다. 인지질에서는 PC함량이 8월 61.2%였으나 4월 50.6%로 줄었고 PS와 PE는 이와 대체로 반대 경향이었다. 중성지질에서는 16 : 0, 16 : 1, 18 : 0, 18 : 1, 20 : 5$({\omega}3)$이 많았고 특히 $20:5({\omega}3)$가 많은 점이 특징적이었으며 월동전 후는 포화산과 monoene산 조성이 줄고 대신 polyene산 조성이 높아지는 경향이었다. 11월은 주로 16 : 0, 16 : 1, 18:1 등 일부가 줄었으나 4월은 16 : 0, 20 : 1, 22 : 1를 제외한 14 : 0, 14 : 1, 16 : 1, 18 : 0, 18:1 등 대부분의 포화 및 monoene산과 18 : 2, 18 : 3$({\omega}3)$이 균일하게 줄어 동면시 이들이 에너지원이 되고 특히 전기간에 걸쳐서는 16 : 1과 18 : 1의 이용이 용이했을 것으로 판단되었다. 인지질에서는 16 : 0, 18 : 0, 20 : 5$({\omega}3)$, 22 : 5$({\omega}3)$, 22 : 6$({\omega}3)$이 많았고 monoene산 조성비는 거의 일정하였으나 월동전 후는 16 : 0, 18 : 0을 주로 한 포화산과 18 : 2, 18 : 3$({\omega}3)$이 줄었고 대신 20 : 4$({\omega}6)$, 20 : 5$({\omega}3)$, 22.5$({\omega}3)$, 22 : 6$({\omega}3)$ 등고도 polyene산 조성이 높아져 불포화도가 증가하였으며, 특히 20 : 4$({\omega}6)$와 20 : 5$({\omega}3)$의 상승폭이 컸다. In order to gain fundamental information on the utilization of lipids as energy source in mud-skipper, muscle lipids and their fatty acid composition were investigated with respect to life cycle-maturation (Aug), before-hibernation (Nov), and after-hibernation (Apr). Crude and neutral lipid were found to decrease from 1.2, 68.3% (Aug) via 0.7, 53.8% (Nov) to 0.4, 42.6% (Apr), respectively, whilst phospholipids and glycolipids increased from 29.5 and 2.2% (Aug) to 52.1 and 5.3% (Apr), respectively. In neutral lipids, TG contents gradually decreased from 53.8% (Aug) via 33.6% (Nov) to 23.1% (Apr), while FFA and sterol contents increased from 13.5 and 14.2% (Aug) to 22.3 and 24.5% (Apr), respectively. In phospholipids, PC content decreased from 61.2% (Aug) to 50.6% (Apr), while changes in PS and PE contents, as a whole, showed the opposite trends. In neutral lipids, the levels of some fatty acids such as 16:0, 16:1, 18:0, 18:1 and 20:5 $({\omega}3)$ were analyzed to be high, with the 20 : 5 being predominant, and the levels of saturated and monoene-acids gradually decreased, while polyene-acids increased in before and after hibernation. In before hibernation, 16:0, 16:1, 18:1 mainly decreased but 18:2, 18:3 $({\omega}3)$ and most of saturated and monoene acids such as 14:0, 14:1, 16:1, 18:0, 18:1 slightly decreased in after hibernation. From these findings, it was suggested that those fatty acids decreased during hibernation were used as a energy source, particularly 16:1 and 18:1 being most preferentially used. In phospholipids, the levels of 16:0, 18:0, 20:5 $({\omega}3)$, 22:5 $({\omega}3)$ and 22:6 $({\omega}3)$ were found to be high. Throughout the life cycle, the levels of monoene-acids in phospholipids stayed constant, whilst those of 18:2, 18:3 $({\omega}3)$ and saturated acids such as 16:0, 18:0 were found to be decreased gradually in before and after hibernation, whereas those for the high degree of polyene-acids such as 20:4 $({\omega}6)$, 20:5 $({\omega}3)$, 22:5 $({\omega}3)$, 22:6 $({\omega}3)$ increased, particularly 20:4 $({\omega}6)$, 20:5 $({\omega}3)$ being most increased.
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