대장균군 검사용 간이 시험지 개발

이인애(In-Ae Lee),김재화(Jae-Wha Kim),이회구(Hee-Gu Lee),성창근(Chang-Keun Seong),최인성(In-Seong Choe),정태화(Tai-Wha Chung) 대한의생명과학회 1996 Biomedical Science Letters Vol.2 No.1

대장균군 검사용 간이 시험지는 본 실험실에서 국내 최초로 고안, 개발하였으며 이 간이 시험지법은 현장 검사법의 하나로 대장균군이 내는 succinic acid dehydrogenase 때문에 tetrazolium salt가 환원되어 적색 반점을 형성하는 것을 이용한 방법으로서 이 간이 시험지의 제조는 대체로 종래의 표준 평판법과 거의 동일한 조성의 배지와 사약을 사용하여 여지에 흡착치킨 후, 건조시켜(60℃) 멸균한 것으로 표준 평판법과 어떤 상관관계가 있는가를 검토하였다. 이 간이 시험지의 제조에서는 bile salt No. 3를 deoxycholate로 대체하여 제조 원가를 절감하였고, 또한 일본에서 현재 시판되고 있는 제품과 품질 비교시험을 하여 더 좋은 결과를 얻었으며 종래의 표준 평판법과 비교하였을 때도 오히려 표준 평판법(24-48시간 배양)보다 빠른 시간(16-20시간 배양)내에 판정할 수 있는 이점이 있으며, 표준 평판법에서는 없어서는 안될 배지나 배양 접시, pipette등의 자료 및 기구가 일체 필요없고 언제 어디서나 현장에서 직접 시험할 수 있어 매우 간편하며 또한 저렴한 가격으로 제조할 수 있는 경제성이 높은 이점을 갖고 있다. The objective of this study was to develop a paper strip which could determine E. coli qualitatively and quantitatively in water, wastewater, drinks, or food. This paper strip method was a simple and rapid test method that determine E. coli by visual identification. In this study, nutrient culture media were formulated and characterized for optimum conditions. Paper strips were then prepared by impregnating into the media and dried at 60℃. The test procedure is quite simple to use. The paper strip was dipped into a sample, and excess sample was removed. The strip was then incubated at 37℃ for 16 to 20 hours and the number of colonies on the strip was counted. The color of the colony spots produced by microorganisms varied depending on the media formulation. Violet-red spots were produced by E. coli. The test method was simple, rapid and no special laboratory equipment was necessary for visual identification. Therefore, this test method is applicable to on-site tests such as field tests or home tests. The paper strip method was compared with the standard agar plate method and Japanese commercial product. The method of the economical preparation of test strips was studied for production on industrial scale.

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 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.

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.

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.

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.

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.

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.

B형 간염 바이러스 단백질에 있어서 HLA-A2에 의해 표현되는 Epitope 펩타이드 들의 분석

이희구,임종석,김승목,이기영,김희수,김승호,권태종,최인성,정태화,김길현 이화여자대학교 생명과학연구소 1995 생명과학연구논문집 Vol.6 No.-

The cytotoxic T lymphocyte(CTL) are an important component in host defense mechanism against viral infection. They can recongnize virus-derived peptides presented by the ClassⅠ MHC molecule at the cell surface of the infected cells. On searching for effective CTL epitopes of hepatitis B virus(HBV), we synthesized a distinct set of 9-10 mer peptide containing amino acid sequence of hepatitis B virus surface proteion that are selected on the basis of a computer modeling and the previously described HLA-A2 specific motifs. Binding assay of the synthetic peptides to HLA-A2 molecules using human antigen processing defectantn T2 cells showed what 3 out of 4 synthetic peptides enhaced the expression of HLA-A2 molemule on T2 cell surface. Two anchor positions, namely P2 and P9(or P10) appeared to play a decisive role for binding. Structural chacteristics of the peptides addressed by molecular dynamics simulation was analysed and compared. These peptides also parially triggerd CTL isolatied frmo human peripheral blood mononuclear cells of HBV positive patients, and the response was peptide-specific. These results showed that negatively-charged amino acid residue at P2 hampered binding affinity of the peptides to HLA-A2 molecules, and that binding affinity of the peptides are not always reflected by thier immunogenicity among natural T cell repertoire.

B형 간염 바이러스 단백질에 있어서 HLA-A2에 의해 표현되는 Epitope 펩타이드 들의 분석

이희구,임종석,김승목,이기영,김희수,김승호,권태종,최인성,정태화,김길현 梨花女子大學校 藥學硏究所 1995 藥學硏究論文集 Vol.- No.5

The cytotoxic T lymphocyte (CTL) are an important component in host defense mechanism against viral infection. They can recongnize virus-derived peptides presented by the Class I MHC molecule at the cell surface of the infected cells. On searching for effective CTL epitopes of hepatitis B virus(HBV), we synthesized a distinct set of 9-10 mer peptide containing amino acid sequence of hepatitis B virus surface protein that are selected on the basis of a computer modeling and the previously described HLA-A2 specific motifs.Binding assay of the synthetic peptides to HLA-A2 molecules using human antigen processing defectant T2 cells showed that 3 out of 4 synthetic peptides enhanced the expression of HLA-A2 molecule on T2 cell surface.Two anchor positions, namely P2 and P9(or P10) appeared to play a decisive role for binding.Structural. characteristics of the peptides addressed by molecular dynamics simulation was analysed and compared.These peptides also partially triggered CTL isolated from human peripheral blood mononuclear cells of HBV positive patients, and the response was peptide-spcific.These results showed that negatively-charged amino acid residue at P2 hampered binding affinity of the peptides to HLA-A2 molecules, and that binding affinity of the peptides are not always reflected by their immunogenicity among natural T cell repertoire.