A Simplified Procedure for C-reactive Protein Purification

이희구,김용호,이홍수,최명자,최인성,정태화,Lee, Hee-Gu,Kim, Yong-Ho,Lee, Hong-Soo,Choi, Myung-Ja,Choe, In-Seong,Chung, Tae-Wha 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.4

환자의 혈장이나 복수로부터 C-reactive protein을 높은 수율로서 순수하게 분리정제할 수 있는 간이 정제법을 개발하였다. DPPC-Affinity chromatography와 Hydroxylapatite chromatography 분리과정을 거쳐 간단하고 신속한 방법으로 분리된 CRP는 단일확산 침전법과 폐렴균의 세포막인 C-polysaccharide를 이용한 EIA 방법으로 활성도를 측정하였다. 분리한 CRP는 SDS-PAGE에서 단일밴드로 나타났으며 immunoelectrophoresis에서도 단일 침전 arc로 나타나 순수분리 되었음을 확인하였다. 또한, Sephacryl S-200 gel filtration과 SDS-polyacrylamide gel 전기영동 분석결과에 의하면 분자량은 118,000으로서 5개의 동일한 subunit로 구성되어 있으며 그 subunit의외 분자량은 23,600이었다. CRP의 isoelectric point는 4.82였으며 500 ml의 복수로부터 분리된 CRP의 회수율은 약 67%로서 4.7 mg이었다. A simplified procedure for obtaining high purity and high yield of C-reactive protein (CRP) was established. The CRP from ascitic and pleural fluid was purified using calcium dependent affinity chromatography of CNBr-activated Sepharose-4B covalently coupled to p-diazonium phenylphosphorylcholine (DPPC) and hydroxylapatite chromatography. The active fractions of CRP during the purification procedure were determined by EIA using pneumococcal C-polysaccharide. The final recovery of CRP was 67% of the initial protein amount as quantitated by single radial immunodiffusion (SRID), and the purification fold was 3,600. The purified CRP was shown to be homogeneous by electrophoresis and immunoelectrophoresis. The molecular weight of CRP was approximately 118KD as determined by Sephacryl S-200 gel filtration and the molecular weight of a subunit was approximately 23.6 KD by SDS-polyacrylamide gel electrophoresis. The isoelectric point of CRP was found to range about 4.82.

C - reactive protein 의 간이 정제법

이희구,김용호,이홍수,최명자,최인성,정태화 ( Hee Gu Lee,Yong Ho Kim,Hong Soo Lee,Myung Ja Choi,In Seong Choe,Tae Wha Chung ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.4

A simplified procedure for obtaining high purity and high yield of C-reactive protein (CRP) was established. The CRP from ascitic and pleural fluid was purified using calcium dependent affinity chromatography of CNBr-activated Sepharose-4B covalently coupled to p-diazonium phenylphosphorylcholine (DPPC) and hydroxylapatite chromatography. The active fractions of CRP during the purification procedure were determined by EIA using pneumococcal C-polysaccharide. The final recovery of CRP was 67% of the initial protein amount as quantitated by single radial immunodiffusion (SRID), and the purification fold was 3,600. The purified CRP was shown to be homogeneous by electrophoresis and immunoelectrophoresis. The molecular weight of CRP was approximately 118KD as determined by Sephacryl S-200 gel filtration and the molecular weight of a subunit was approximately 23.6 KD by SDS-polyacrylamide gel electrophoresis. The isoelectric point of CRP was found to range about 4.82.

인체내 혈청 중의 보체 C3 의 정제 및 특성연구

이희구,이종순,최명자,최인성,정태화 ( Hee Gu Lee,Jong Soon Lee,Myung Ja Choi,In Seomg Choe,Tai Wha Chung ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.1

The third component of complements (C3) has been purified from plasma (blood type: B) by sequential employment of QAE-Sephadex A-50 chromatography, 20% (w/v) PEG-3,350 precipitation, heparin-Sepharose CL-6B, and hydroxylapatite chromatographies. Final recovery of C3 was 57% as quantitated by single radial immunodiffusion (SRID), and purification fold was 78. The SDS-PAGE indicated that the purified C3 was homogeneous. Mercury ions or low concentration of magnesium ion showed stimulatory effects on the hemolytic activity of C3, which were inhibited by EDTA or high concentration of reducing agent. Degradation of C3 appeared to be selectively enhanced by several factors, while lysate of sheep red blood cells (SRBC) inhibited it.

Anti-Progesterone Monoclonal Antibody에 대한 Anti-Idiotype 항체의 불임효과에 관한 연구

윤도영,남경수,이희구,이홍수,최명자,최인성,김종배,정태화,Yoon, Do-Young,Nam, Kyung-Soo,Lee, Hee-Gu,Lee, Hong-Soo,Choi, Myung-Ja,Choe, In-Seong,Kim, Jong-Bae,Chung, Tai-Wha 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.6

프로제스테론에 대한 단일클론항체(15A)를 항원으로 토끼의 anti-idiotypic antibody(anti-Id)를 생산한 후 분리 정제하였다. 또한 단일클론성 anti-Id를 분비하는 세포주를 개발하기 위해 15A를 Balb/c 생쥐에 면역주사 하였다. 그 후 항체를 생산하는 B lymphocyte와 myeloma 세포를 융합시켜 하이브리도마 세포를 만든 뒤 이들 중 anti-Id를 생산하는 3종류의 클론(G2, G9, H6)을 선별하였다. Anti-Id의 효소 면역측정법은 horseradish peroxidase(HRP)를 활성화시킨 후, 15A와 반응시켜 만든 15A-HRP를 사용하였다. Anti-Id의 불임에 미치는 효과를 control과 비교하여 본 결과, control로서 anti-mouse IgG를 투여한 그룹은 임선율이 86.7%로 높게 나타났으나, progesterone-BSA를 투여한 후 그룹은 임신율이 30.4%로 임신이 억제되었고, anti-Id를 주사한 그룹은 임신율이 45.5%이었다. 이 때 각 그룹에 속한 개체들의 프로제스테론에 대한 항체농도를 측정한 결과는 각각 $0.25{\mu}g/ml$, $3,500{\pm}950{\mu}g/ml$, 그리고 $31.3{\pm}7.4{\mu}g/ml$로 나타났다. 또한 anti-Id를 주사한 경우 임신한 쥐들의 경우($10.1{\pm}1.3{\mu}g/ml$) 보다도 임신하지 않은 쥐들에서 프로제스테론에 대한 항체 농도가 $43.8{\pm}6.1{\mu}g/ml$로 4배가량 높게 나타났다. 이와 같은 결과에서 anti-Id를 주사한 쥐들의 그룹에서 프로제스테론에 대한 항체의 농도와 임신억제 효과는 상관관계가 있음을 알 수 있었다. 본 실험의 결과를 종합해 볼 때 anti-Id를 주사한 그룹의 쥐들이 progesterone-BSA를 능동면역한 그룹의 쥐들보다 임신율은 높게 나타났으나, control 그룹의 임신율 86.7%와 비교할 때 현저한 피임효과를 나타냄으로써 anti-Id를 피임백신으로 사용할 수 있는 가능성을 보여 주었다. The active immunization of progesterone produces high titer antibodies. However, the progesterone itself binds to the receptor sites and produces the side effect on the contraceptive activities of the antibodies. Therefore, the anti-idiotypic antibody (anti-Id) was studied whether it had the same effect as the active immunization of progesterone to prevent pregnancy or not. If it has antifertility effect, it could be utilized as a contraceptive vaccine. The passive immunization of antibody to mice was previously studied and found that anti-progesterone monoclonal antibody prevents pregnancy by degenerating the embryos in the oviduct and uterus (Yoon et al., 1989). The polyclonal anti-Id was obtained by immunizing anti-progesterone monoclonal antibody (15A) to rabbit and then purified using affinity column chromatography. Furthermore, three different clones (G2, G9, H6) of monoclonal anti-Ids have been raised against anti-progesterone monoclonal antibody. The relationships between the antifertility effects and anti-progesterone antibody level were studied by the active immunization of anti-Id and progesterone-BSA. The results showed that a higher antifertility effect was observed with progesterone-BSA immunization (70%) than with anti-Id immunization (55%). The plasma level of anti-progesterone was as high as $3,500{\mu}g/ml$ with progesterone-BSA immunization. The comparison study of the antibody levels between pregnant ($10{\mu}g/ml$) and non-pregnant mice ($44{\mu}g/ml$) after immunization with the anti-Id indicates that antibody formation at a certain level prevents pregnancy. However, the antibody level is not proportional to the antifertility effect in mice.

Anti - Progesterone Monoclonal Antibody 에 대한 Anti - Idiotype 항체의 불임효과에 관한 연구

윤도영,남경수,이희구,이홍수,최명자,최인성,김종배,정태화 ( Do Young Yoon,Kyung Soo Nam,Hee Gu Lee,Hong Soo Lee,Myung Ja Choi,In Seong Choe,Jong Bae Kim,Tai Wha Chung ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6

The active immunization of progesterone produces high titer antibodies. However, the progesterone itself binds to the receptor sites and produces the side effect on the contraceptive activities of the antibodies. Therefore, the anti-idiotypic antibody (anti-Id) was studied whether it had the same effect as the active immunization of progesterone to prevent pregnancy or not. If it has antifertility effect, it could be utilized as a contraceptive vaccine. The passive immunization of antibody to mice was previously studied and found that anti-progesterone monoclonal antibody prevents pregnancy by degenerating the embryos in the oviduct and uterus (Yoon et al., 1989). The polyclonal anti-Id was obtained by immunizing anti-progesterone monoclonal antibody (15A) to rabbit and then purified using affinity column chromatography. Furthermore, three different clones (G2, G9, H6) of monoclonal anti-Ids have been raised against anti-progesterone monoclonal antibody. The relationships between the antifertility effects and anti-progesterone antibody level were studied by the active immunization of anti-Id and progesterone-BSA. The results showed that a higher antifertility effect was observed with progesterone-BSA immunization (70%) than with anti-Id immunization (55%). The plasma level of anti-progesterone was as high as 3,500 ㎍/㎖ with progesterone-BSA immunization. The comparison study of the antibody levels between pregnant (10 ㎍/㎖) and non-pregnant mice (44 ㎍/㎖) after immunization with the anti-Id indicates that antibody formation at a certain level prevents pregnancy. However, the antibody level is not proportional to the antifertility effect in mice.

인형극 활동이 정신지체 아동의 수용언어 및 표현언어에 미치는 영향

국미경(Kuk Mi-Kyong),이희구(Lee Hee-Gu) 한국재활과학회 2004 難聽과 言語障碍 Vol.27 No.1

The purpose of this study was to examine the effect of doll play on the receptive language and expressive language of mentally retarded children. The subjects in this study were three selected mentally retarded children from special school. After the doll play physical activity and doll-making program were conducted in 10 sessions, the main doll-play program was implemented in 40 sessions. The findings of this study were as below: First, the verbal guidance program by doll play had an effect on enhancing the receptive language skills of the mentally retarded children. Though there was an individual difference between the three children in receptive language age, social age, cognitive ability and environment, Child 1 made a great progress in sentence comprehension, part of receptive language skills in which he was particularly interior, compared to rich expression skills. Child 2 also improved after the experiment, though his change wasn't so noticeable, compared to the other children. Child 3 showed the best change in the comprehension of sentence, tense, adjective, and interrogative. Second, the verbal guidance program by doll play had an effect on developing the expressive language skills of the mentally retarded children. The pretest and posttest showed that Child 1 made the greatest change in every area. Child 2 didn't show much improvement due to poorer cognitive ability and vocabulary skills, but he used subject and verb much more often than in the past that just a ,"vord or a syllable was used. Child 3. didn't make much progress in verbal skills, compared to his cognitive ability and verbal comprehension, but his use of logical and accurate expressions increased a lot in speaking with three syllables or about past experiences. As just three mentally retarded children were investigated as a case study, the findings of this study might not be generalizable. There are some suggestions based on the above-mentioned findings: First, exceptional children 'who lack attention span and learning spontaneity could be helped by doll play to be fully motivated and interactive in class. Therefore, it's needed to develop doll-play teaching programs tailored to different types of instruction and subjects and to provide organized, systematic teacher training. Second, as it's found in this study that doll play not only gives a positive impact on the receptive and expressive language of children but brings other areas change, the impact of doll play on the sociability, confidence and human relations of children should be studied deeply.

쥐 간세포 일차배양 세포의 생존능과 대사능에 단층과 복층 콜라젠 젤이 미치는 영향의 비교

정미경(Mi-Kyung Chung),이혜경(Hae Kyung Lee),이희구(Hee Gu Lee),양은경(Eun Kyung Yang),박정극(Jung Keug Park),이태규(Tae Gyu Lee),임종순(Jong Soon Lim),박순희(Sue Nie Park) 한국생물공학회 1996 KSBB Journal Vol.11 No.4

Vinblastine Determination Measured by a Sensitive ELISA Inhibition Assay

Jae-Wha Kim(김재화),Mi-Young Han(한미영),Hee-Gu Lee(이희구),Eun-Young Song(송은영),Tai-Wha Chung(정태화),Kyung-Soo Nam(남경수),In-Seong Choe(최인성),최용경 대한의생명과학회 1996 Biomedical Science Letters Vol.2 No.1

Vinblastine을 포함하는 bis-indole alkaloids에 대한 단일클론 항체를 생산하여 Vinca alkaloids의 양을 측정할 수 있는 간편한 immunoassay 체계를 확립하였다. Vinca alkaloids는 periwinkle식물체의 배양된 세포로부터 추출하여 BSA와 접합한 후 Balb/c생쥐에 면역시켜 얻은 비장세포와 골수종양세포의 융합을 유도하여 VBL-BSA에 반응하는 클론을 ELISA 방법으로 분석하였으며 이들 클론 중 bis-indole alkaloids와 특이적으로 반응하는 항체는 inhibition assay를 통하여 분리할 수 있었고 그 결과 두 개의 단일클론 항체를 형성하는 세포주(KN-1과 KN-2)를 확립하였다. KN-1의 경우 dimeric bis-indole alkaloids와는 상당한 교차반응을 나타낸 반면 monomeric bis-indole alkaloids와는 교차반응을 나타내지 않았으며 이 클론의 항체를 이용하여 배양된 세포 추출물에 포함된 Vinca alkaloids의 양을 측정한 결과 0.05 nM정도의 dimeric Vinca alkoloids까지도 측정할 수 있었다. Specific monoclonal antibodies(mAbs) against bis-indole alkaloids related to vinblastine were established to develop a simple and specific immunoassay system for the quantitation of Vinca alkaloids. Vinca alkaloids were extracted from tissue cultured cells of periwinkle plant (Vinca rosea L.). Spleen cells from Balb/c mice immunized with vinblastine-bovine serum albumin(VBL-BSA) conjugate as immunogen were fused with myeloma cells(Sp2/0-Ag.14) in the presence of polyethylene glycol. In the preliminary experiments, 32 clones which highly reacted with VBL-BSA conjugate were selected by ELISA(Enzyme-linked immunosorbent assay). These clones were further analyzed by inhibition assay of ELISA. The results obtained with two typical monoclonal antibodies, KN-1 and KN-2, were described. KN-1 exhibited considerable reactivities with soluble dimeric bis-indole alkaloids, whereas no cross reacted with monomeric bis-indole alkaloids. However KN-2 showed cross reactivity with mono- and di-meric bis-indole alkaloids. Furthermore, KN-1 was applied to the immunoassay system for determining the VBL amounts of in vitro cultured cell extracts. This assay system could detect dimeric vinca alkaloid as low as 0.05 nM.

미립자 응집반응을 이용한 C-reactive Protein의 면역측정법에 관한 연구

김재화(Jae-Wha Kim),송은영(Eun-Young Song),이희구(Hee-Gu Lee),최용경(Yong-Kyung Choe),최명자(Myung-Ja Choi),김용호(Yong Ho Kim),최인성(In-Seong Choe),정태화(Tai-Wha Chung) 대한의생명과학회 1996 Biomedical Science Letters Vol.2 No.1

환자의 복수와 늑막액으로부터 p-diazonium phenylphosphorylcholine(DPPC) coupled Separose-4B affinity chromatography와 hydroxylapatite chromatography를 실시하여 C-reactive protein(CRP)를 분리, 제정하였다. 정제된 CRP를 토끼에게 면역화하여 항혈청을 얻고 affinity chromatography를 하여 면역항체(IgG)를 분리하였다. 분리된 면역항체를 미립자에 감작시킨 후 미립자 응집반응에 의하여 3분내에 CRP를 측정할 수 있는 간이 면역측정법을 개발하였다. 본 연구에서 개발된 CRP측정법의 검출범위는 0.5~20㎎/dl이며, 임상 시험 결과 0.7~2.9㎎/dl에서는 강한 응집반응을, 5.0~13.2㎎/dl에서는 약한 응집반응을 보였고 28㎎/dl이상에서는 항원 과잉으로 인한(zone of Ag excess phenomenon) 위음성을 나타냈다. 74명의 환자 혈청을 대상으로 CRP의 농도를 조사한 결과 평균치는 3.8㎎/dl이었으며 대부분의 환자에서는 10㎎/dl 이하의 농도로 존재하였다. 그러므로 1차판정시 음성을 나타낸 시료라도 혈청을 5~10배정도 희석하여 재분석한다면 오차없이 CRP를 검출할 수 있었다. 환자 혈청을 검체로 하여 본 연구에서 개발한 면역측정법과 현재 수입 시판중인 프랑스의 B사 제품과 일본의 I사 제품을 비교한 결과 좋은 상관관계를 보였다. 이와 같은 평가분석을 통하여 볼 때 본 연구에서 개발한 간이 면역측정법은 사용이 비교적 간편하며 신빙성이 있어 CRP를 스크리닝 하는데 효과적임을 알 수 있었다. The C-reactive protein(CRP) from ascitic and pleural fluid was purified using calcium dependent affinity chromatography of CNBr activated Sepharose-4B covalently coupled to p-diazonium phenylphosphorylcholine(DPPC) and hydroxylapitite chromatography. Polyclonal antibody was prepared from rabbit by immunizing the purified CRP. Specific immunoglobulin G was isolated using affinity chromatography and coupled to microparticles. A sensitive microparticle-based immunoassay was developed to measure CRP within 3 mins. The detection range was between 0.5㎎/dl and 20㎎/dl in serum, showing strong response in the range of 0.7~2.9 ㎎/dl, weak response in 5.0~13.2 ㎎/dl and zone phenomenon over 28㎎/dl. The average value of CRP in 74 samples was 3.8㎎/dl and most of the values were lower than 10㎎/dl. The CRP values of serum samples were determined by our microparticle-based immunoassay, and were compared with those obtained using the other commercial products(B Co., France and I Co., Japan). Good correlations were shown between the values obtained by our developed mi-croparticle-based immunoassay system and those by other commercial products. All performance characteristics evaluated make our developed microparticles-based immunoassay suitable for a simple, rapid, and reliable screening of CRP in serum.

항바이러스 활성 식물자원 탐색

권두한(Dur Han Kwon),김만배(Man Bae Kim),윤도영(Do Young Yoon),이영희(Young Hee Lee),김재화(Jae Wha Kim),이희구(Hee Gu Lee),최인성(In Seong Choi),임종석(Jong Seok Lim),최용경(Yong Kyeung Choe) 한국약용작물학회 2003 한국약용작물학회지 Vol.11 No.1

Aqueous extracts form eleven species of Korean medicinal plants were screened for antiviral activity against transmissble gastroenteritis virus (TGEV) and porcine epidemic diarrhae virus (PEDV). Seven of eleven plant extracts were found to have cytotoxicity for ST cells at the concentration of 1㎎/ml, and ten extracts except Ephedra sinica were found to have non-cytotoxicity for Vero cells at the same concentration. Extracts of Znthoxylum piperium, Cudrina tricuspidata, Clerodendron trichomum, Sophora flavescens, effectively inhibited TGEV, respectively. There were the antiviral activity aganist TGEV in the extracts of Glesdistsia sinensis, Euonymus alatus, Ficus carica, Solanum nigrum, Ephodra sinica, Xanthium strumarium, and Acanthopanax seeliflorus in spite of their cytotoxicities. Nine extracts except Xanthium strumarium and Acanthopanax seeliflorus were found to have the antiviral activitity against PEDV. Extracts of Zanthoxylum piperium, Sophora flavescens was shown to have antiviral activities against both TGEV and PEDV.