Production, Purification and Characterization of Monoclonal Antibodies Specific to Progesterone

윤도영,최상훈,최명자,최인성,정태화,Yoon, Do-Young,Choi, Sang-Hoon,Choi, Myung-Ja,Choe, In-Seong,Chung, Tai-Wha Korean Society for Biochemistry and Molecular Biol 1991 한국생화학회지 Vol.24 No.1

Progesterone-$11{\alpha}$-hemisuccinate-BSA를 제조하여 Balb/c 생쥐에 주사하여 얻은 비장세포를 myeloma 세포와 융합하여 여러 가지 subtype 형태($IgG_1$, $IgG_{2a}$, $IgG_{2b}$, IgM)를 가진 항체를 분비하는 7개의 하이브리도마 세포주를 얻었다. 이들이 분비하는 항체들을 간단히 ammonium sulfate 침전법, Protein A-Sepharose 4B 등으로 분리정제하여 그 특성을 조사하였다. 그 결과 단일클론 항체가 18-44% 정도의 수률로 SDS-PAGE상에서 순수분리 되었음을 확인하였다. 이들 항체들의 affinity constant는 $5{\times}10^7-4{\times}10^8M^{-1}$ 정도였으며, 이들 항체들은 Progesterone-3-(O-carboxymethyloxime)-BSA를 면역원으로 하여 얻은 항체와 비교했을 때, 특이성과 다른 steroid 호르몬과 교차반응성 등이 클론에 따라 특이하게 나타났다. 특히 이들 항체 중 051-01 clone은 다른 clone에 비해 매우 빠른 성장을 보였으며, 항체의 양이 복수액의 전체 단백질의 38% 정도나 되어 titer level이 매우 높았으며, 방사면역측정법이나 효소면역측정법으로 반응성을 측정했을 때 그 감도가 매우 좋았다. Seven hybridoma cell lines, 051-01 ($IgG_1$), 051-02 ($IgG_{2b}$), 051-03 ($IgG_1$), 051-04 (IgM), 051-05 ($IgG_{2a}$), 051-06 (IgM), and 051-07 ($IgG_{2a}$) prodcing monoclonal antibodies against progesterone were established. Progesterone $11{\alpha}$-hemisuccinate-bovine serum albumin conjugate was prepared using carbodiimide and then used as an immunogen. Spleen cells of Balb/c mice immunized with the immunogen were fused with mouse myeloma (P3-X63-Ag8.653) cells. Hybridoma cells were screened by radioimmunoassay using $^3H$-progesterone. The clones 051-01, 051-03, and 051-05 were purified using protein A-Sepharose 4B affinity chromatography utilizing their subtype characteristics. The purity of the isolated IgG was identified by SDS-PAGE. The progesterone binding capacity of the 5 monoclonal antibodies ranged from 7 to 72 pmole/mg IgG and the affinity constant ranged from $5.0{\times}10^7\;M^{-1}$ to $4{\times}10^8\;M^{-1}$ by the Scatchard analysis method. Cross-reactivities of the monoclonal antibodies, 051-01 and 051-02 with other steroid hormones were compared. These antibody reactions were very specific to progesterone and sensitive in a competitive immunoassay.

프로제스테론 단일클론 항체의 생산과 정제 및 특성연구

윤도영,최상훈,최명자,최인성,정태화 ( Do Young Yoon,Sang Hoon Choi,Myung Ja Choi,In Seong Choe,Tai Wha Chung ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.1

Seven hybridoma cell lines, 051-01 (IgG₁), 051-02 (IgG_(2b)), 051-03 (IgG₁,), 051-04 (IgM), 051-05 (IgG_(2a)), 051-06 (IgM), and 051-07 (IgG_(2a)) prodcing monoclonal antibodies against progesterone were established. Progesterone llα-hemisuccinate-bovine serum albumin conjugate was prepared using carbodiimide and then used as an immunogen. Spleen cells of Balb/c mice immunized with the immunogen were fused with mouse myeloma (P3-X63-Ag8.653) cells. Hybridoma cells were screened by radioimmunoassay using ³H-progesterone. The clones 051-01, 051-03, and 051-05 were purified using protein A-Sepharose 4B affinity chromatography utilizing their subtype characteristics. The purity of the isolated IgG was identified by SDS-PAGE. The progesterone binding capacity of the 5 monoclonal antibodies ranged from 7 to 72 pmole/㎎ IgG and the affinity constant ranged from 5.0×10^7 M^(-1) to 4×10^8 M^(-1) by the Scatchard analysis method. Cross-reactivities of the monoclonal antibodies, 051-01 and 051-02 with other steroid hormones were compared. These antibody reactions were very specific to progesterone and sensitive in a competitive immunoassay.

Creatine Kinase Isoenzyme 의 분리와 정제에 관한 연구

권두한,윤도영,이홍수,최명자,최인성,정태화 ( Dur Han Kwon,Do Young Yoon,Hong Soo Lee,Myung Ja Choi,In Seong Choe,Tai Wha Chung ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.4

Bovine creatine kinase MM was purified from bovine heart muscle, through homogenization, ethanol precipitation, DEAE-cellulose chromatography, two step of gel filtration chromatography and affinity chromatography, Human CK-MM wa spurified from fetal human heart muscle through homogenization, ethanol precipitation, and DEAE-cellulose chromatography. Specific activities of bovine and human CK-MM were presented at an activity of 801 U/㎎ and 369 U/㎎, respectively. CK-BB from bovine brain and human fetal brain tissue were purified by homogenization, ethanol precipitation and DEAE-cellulose chromatography. Specific activities of bovine and human CK-BB appeared to 702 U/㎎ and 615 U/㎎, respectively. Ck-MB isoenzyme was synthesized by recombining each single subunit of MM and BB which were prepared by treating with 8 M guanidine-HCl, and its activity was identified on agarose gel electrophoresis.

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.

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.

IL-1 수용체를 통한 T세포 활성화에 Genistein 과 Staurosporin이 미치는 영향

한미영,윤도영,오은숙,고우석,윤선영,정태화 大韓免疫學會 1996 大韓免疫學會誌 Vol.18 No.4

Stimulation of T-cells through TCR/CD3 complex alone is known to be not enough but also require a costimulatory signal, such as IL-1 stimulation. to induce IL-2 production. In this report, we studied the differential effects of staurosporin (a PKC inhibitor) and genistein (a PTK inhibitor) on the IL-2 production by EL-4 and A2.5 cells, to determine which pathway is responsible for IL-1 receptor signal transduction. While staurosporin suppressed the IL-2 production induced in EL-4 cells upon PMA stimulation but not upon PHA plus IL-1, genistein inhibited the IL-2 production induced by PHA plus IL-1 but not by PMA. Genistein also completey abrogated the IL-2 production by A2.5 cell when stimulated with PHA and IL-1. These results suggest that the costimulatory signal by IL-1, which leads to IL-2 production and proliferation, is closely linked to the PTK activity in T cells.

Immunobiochemical Analysis of the Binding Pocket of Monoclonal Anti-progesterone Antibodies

Yoon, Do Young,Kim, Jae Wha,Lim, Jong-Seok,Choi, Myung Ja,Choe, Yong-Kyung,Kim, Kilhyoun,Choe, In Seong,Chung, Tai Wha 梨花女子大學校 藥學硏究所 1995 藥學硏究論文集 Vol.- No.5

Carrier protein을 progesterone의 C-ring(C-11 위치)에 결합시켜 progesterone에 특이적인 단일크론항체 (mAb)를 만들고 이 항체와 tracer로서 progestorone의 A-ring(C-3 위치)에 horseradish peroxidase를 결합시킨 progesterone-3 (O-carboxy methyloxime)-horseradish peroxidase를 이용하여, 혈청 중의 콜레스테롤 결합단백질을 불활성화시키는 시약을 tracer에 첨가하여 스테로이드 홀몬을 유기용매로 추출하는 과정없이 혈청 중의 progesterone을 직접 측정하는 효소면역 측정법을 개발하였다. 이 효소면역 측정법을 이용하여 progesterone과 유사한 11개의 스테로이드에 대한 단일크론 항체의 반응특이성을 조사하므로써, progesterone에 대한 단일크론 항체의 항원 반응 부위(antigen binding site)에 스테로이드가 어떻게 결합되어 있는지를 알아 보고자 하였다. 단일크론 항체의 면역화학적 분석결과 스테로이드의 D-ring이 A-ring에 비해 항체의 결합부위(binding pocket)에 훨씬 더 깊이 묻혀 있는 것으로 사료되었다.

Immunobiochemical Analysis of the Binding Pocket of Monoclonal Anti-progesterone Antibodies

Yoon, Do Young,Kim, Jae Wha,Lim, Jong-Seok,Choi, Myung Ja,Choe, Yong-Kyung,Kim, Kilhyoun,Choe, In Seong,Chung, Tai Wha 이화여자대학교 생명과학연구소 1995 생명과학연구논문집 Vol.6 No.-

Carrier protein을 progesterone의 C-ring(C-11 위치)에 결합시켜 progesterone에 특이적인 단일크론항체 (mAb)를 만들고 이 항체와 tracer로서 progesterone의 A-ring (C-3 위치)에 horseradish peroxidase를 결합시킨 progesterone-3(O-carboxy methyloxime)-horseradish peroxidase를 이용하여, 혈청 중의 콜레스테롤 결합단백질을 불활성화시키는 시약을 tracer에 첨가하여 스테로이드 홀몬을 유기용매로 추출하는 과정없이 혈청 중의 progesterone을 직접 측정하는 효소면역 측정법을 개발하였다. 이 효소면역 측정법을 이용하여 progesterone과 유사한 11개의 스테로이드에 대한 단일크론 항체의 반응특이성을 조사하므로써, progesterone에 대한 단일크론 항체의 항원 반응 부위(antigen binding site)에 스테로이드가 어떻게 결합되어 있는지를 알아 보고자 하였다. 단일크론 항체의 면역화학적 분석결과 스테로이드의 D-ring이 A-ring에 비해 항체의 결합부위(binding pocket)에 훨씬 더 깊이 묻혀 있는 것으로 사료되었다.

Streptomyces 배양액에서의 Interleukin-1 유사물질의 동정

남명수,배윤수,윤도영,남경수,최인성,정태화 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.2

Streptomyces의 배앙액이 쥐의 흉선세포(thymocyte)를 활성화시켰는데 그것은 농도에 의존적이었고 receptor binding 분석법에서는 C_3H/HeJ의 흉선세포에 있는 IL-1 receptor에 IL-1β-FITC의 결합을 경쟁적으로 억제하였다. Streptomyces의 배앙액에 있는 단백질을 분리하기 위해서 ultrogel AcA44 column chromatography를 하여 흉선세포의 증식이 제일 많은 분획을 모아서 FPLC용 column인 superose 12에 loading한 결과 흉선세포의 증식이 제일 높은 분획은 21번 분획이었다. Affinity chemical cross-linking 실험에서 미생물 배양액의 활성인자는 IL-1 receptor를 통하여 흉선세포의 활성을 유발하는 것으로 생각된다. 미생물 배양액에서 분리정제된 단백질은 anti-rIL-1β 항체와 반응하며, western blotting 결과 그 분자량은 약 21 kd으로 추정되었다. We have identified a T cell-activating material in the culture supernatant of Streptomyces species. The factor in microbial culture supernatant (MCS) induced thymocyte proliferation in a dose dependent fashion and it could be detected by immunoblot analysis using anti-interleukin1(IL-1) antibody. The factor in MCS was slightly larger(about 21 kd) in its molecular weight than IL-1 on SDS-PAGE. When ^125I-MCS was covalently coupled with homo-bifunctional cross-linking agent, disuccinimidyl-propionate to IL-1 receptor(IL-1R) on mouse thymoma cell(EL-4) and immunoprecipitated with anti-IL-1R antibody the molecular weight of this complex of 110 kd was observed. Interaction between IL-1 receptor and the factor in MCS could be inhibited by addition of excess IL- lβ. These results suggest that Streptomyces species may produce a factor which is closely related to IL-1β in its functional and biochemical characteristics.