누드마우스에 이식된 인체대장암에서 I - 131 표지 항태아성암항원 단일클론항체를 이용한 방사면역치료법 : 치료성적에 관계되는 인자분석

김병태(Byung Tae Kim),고창순(Chang Soon Koh),이명철(Myung Chul Lee),정준기(June Key Chung),김상은(Sang Eun Kim),최용(Yong Choi),이경한(Kyung Han Lee),지대윤(Dae Yoon Chi),정홍근(Hong Keun Chung) 대한핵의학회 1995 핵의학 분자영상 Vol.29 No.3

N/A This study was designed to evaluate the effects of various factors on the therapeutic effect of the I-l3l labeled anti-carcinoembryonic antigen monoclonal antibody(anti-CEA antibody). Tetrazolium-based colorimetric assay (MTT) was used to compare in vitro cytotoxicity of 3 Korean colon cancer cell lines (SNU-C2A, SNU-C4, SNU-C5) for selection of proper 2 cell lines in this study. The changes of the size of tumor which was xenografted to nude mice (balb/c nu/nu) were compared in 4 groups (group treated I-131 labeled anti-CEA antibody, group treated with non-radiolabeled anti-CEA antibody, group treated with I-131. labeled anti-human chorionic gonadotropin monoclonal antibody (anti-hCG antibody) as nonspecific antibody, and group injected with normal saline as a control). Immunohistochemical staining and in vivo autoradiography were performed after excision of the xenografted tumor. The results were as below mentioned. The in vitro cytotoxic effect of I-131 labeled anti-CEA antibody is most prominent in SNU-C5 cell line between 3 cancer cell lines. The changes of xenografted tumor size in both SNU-C4 and SNU-C5 cell tumors at the thirteenth day after injection of the antibodies were smallest in the group treated with I-131 labeled anti-CEA antibody (SNU-C4/SNU-C5; 324/342%) comparing with other groups, group treated with anti-CEA antibody (622/660%), group treated with I-131 anti-hCG antibody (538/546%), and control group(1030/724%) (p〈0.02 in SNU-C4 and p〈0.1in SNU-C5 at the 13th day after injection of antibodies). On the thirteenth day after injection of the antibodies nude mice were sacreficed to count the radiouptake of tumor and to check the changes of tumor size. Correlations between radiouptake and change of tumor size were calculated in each groups and significant negative correlation was only obtained in the group treated with I-131 anti-CEA antibody (p〈0.05). There were no correlations between antigenic expression of carcinoembryonic antigen and distribution of anti-CEA antibody in both SNU-C4 and SNU-C5-cell tumors on immunoperoxidase staining Qn in vivo autoradiography the distributions of anti-CEA antibody were heterogeneous and the intensities of binding were various in SNU-C4 and SNU-C5 cell tumors. It is concluded that I-131 labeled tumor-specific monoclonal antibody, anti-CEA antibody is effective in suppressing the xenografted tumor growth and the effect is influenced by sensitivity of tumor cell itself to the radiolabeled antibody and other local factors instead of specificity of antibody.

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.

소 전염성비기관염(傳染性鼻氣管炎) 바이러스에 대한 monoclonal antibody 생산(生産)과 진단법(診斷法) 개발 II. Monoclonal antibody를 이용한 소 전염성비기관염(傳染性鼻氣管炎)의 진단(診斷)

전무형,김덕환,안수환,이중복,민원기,Jun, Moo-hyung,Kim, Duck-hwan,An, Soo-hwan,Lee, Jung-bok,Min, Won-gi 대한수의학회 1989 大韓獸醫學會誌 Vol.29 No.1

To develop more specific and sensitive diagnostic methods for infectious bovine rhinotracheitis, 7-C-2 monoclonal antibody specific to polypeptides of infectious bovine rhinotracheitis virus (IBRV) was applied in indirect immunofluorescence antibody assay (IFA), indirect immunoperoxidase assay(IPA) and radial immunodiffusion enzyme assay (RIDEA). It was found that IBRV infected in MDBK cells could be detected as early as 8 hours post infection by IFA, and that IFA was more rapid and specific to identify IBRV antigen than IPA. The diagnostic efficacy of RIDEA and SN test was studied with 88 bovine sera. It was evident that RIDEA could eliminate the false positive reaction encountered in serum neutralization(SN) test, being more rapid and sensitive than the latter. Highly significant correlation coefficiency (r=0.76, p<0.01) was evaluated between the titers of sera and the diameters of RIDEA. Tracheal membranes and sera collected from 96 slaughtered cattle with lesions in respiratory organs were examined to detect IBRV antigen and antibody by IFA, RIDEA and SN test. It was presented that positive rates were 32.3% in IFA, 20.8% in RIDEA and 21.9% in SN test, and that coincidence rate between RIDEA and SN test were 100% in positive sera and 98.7% in negative sera. In conclusion, it was assumed that application of monoclonal antibody could improve the diagnostic efficacy of IBR by enhancing sensitivity and specificity of IPA, IFA and RIDEA.

소 傳染性鼻氣管炎 바이러스에 대한 monoclonal antibody 生産과 診斷法 開發 : Ⅱ. Monoclonal antibody를 이용한 소 傳染性鼻氣管炎의 診斷 Ⅱ. Diagnosis of infectious bovine rhinotracheitis by using monoclonal antibody

全茂炯,金德煥,安壽煥,李重馥,閔元其 충남대학교부설 생명공학연구소 1991 생물공학연구지 Vol.1 No.-

To develop more specific and sensitive diagnostic methods for infectious bovine rhinotracheitis, 7-C-2 monoclonal antibody specific to polypeptides of infectious bovine rhinotracheitis virus (IBRV) was applied in indirect immunofluorescence antibody assay(IFA), indirect immunoperoxidase assay(IPA) and radial immunodiffusion enzyme assay (RIDEA). It was found that IBRV infected in MDBK cells could be detected as early as 8 hours post infection by IFA, and that IFA was more rapid and specific to identify IBRV antigen than IPA. The diagnostic efficacy of RIDEA and SN test was studied with 88 bovine sera. It was evident that RIDEA could eliminate the false positive reaction encountered in serum neutralization(SN) test, being more rapid and sensitive than the latter. Highly significant correlation coefficiency(r=0.76, p<0.01) was evaluated between the titers of sera and the diameters of RIDEA. Tracheal membranes and sera collected from 96 slaughtered cattle with lesions in respiratory organs were examined to detect IBRV antigen and antibody by IFA, RIDEA and SN test. It was presented that positive rates were 32.3% in IFA, 20.8% in RIDEA and 21.9% in SN test, and that coincidence rate between RIDEA and SN test were 100% in positive sera and 98.7% in negative sera. In conclusion, it was assumed that application of monoclonal antibody could improve the diagnostic efficacy of IBR by enhancing sensitivity and specificity of IPA, IFA and RIDEA.

Production of a Monoclonal Antibody to Human α-Fetoprotein and Development of Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assays for Human α-Fetoprotein

Yoon,Michung,Lee,Hyung-Hee,Lee,Youngwon THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1999 Journal of biomedical laboratory sciences 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 k 경사슬의 isotype을 나타냈다. 또한 immunoblotting 방법과 ELISA로 특이도를 조사한 결과 모노클로날 항체는 AFP와만 반응하였고, 결합 친화상수는 0.8 ×10 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 k 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 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 immuno-diagnostic kit for the measurement of AFP concentration.

Fast and Simple Qualitative/Semi-Quantitative Analysis of Monoclonal Antibody Mixtures Using Liquid Chromatography–Electrospray Triple Time-of-Flight Mass Spectrometry

변진주,박민호,신석호,신영근 대한화학회 2018 Bulletin of the Korean Chemical Society Vol.39 No.12

Bispecific antibodies are generally prepared by co-expression of mixtures of recombinant monoclonal antibodies. Because of increased molecular complexity, characterization of a desired bispecific antibody or a mixture of monoclonal antibodies is more challenging than characterization of a conventional single monoclonal antibody. The purpose of this study is to develop a fast and simple method for qualitative/semi-quantitative analysis of antibody mixtures using liquid chromatography?electrospray triple time-of-flight mass spectrometry (LC-ESI-TOF/MS) to complement the enzyme-linked immunosorbent assay. To demonstrate the proof of concept for the analysis of antibody mixtures, three different tool monoclonal antibodies (trastuzumab, rituximab, and cetuximab) with various mixture ratios were treated by either PNGase or Fabricator to simplify the antibody structures without glycans. After deglycosylation, the mixtures of antibodies were analyzed by LC-ESI-TOF/MS in the positive ion mode. The m/z scan range of 2000?4000 was used for the deconvolution of each peak from antibodies. Because each antibody could show different ionization efficiency in TOF MS, the peak intensities obtained from various mixture antibodies (1:6:3 or 3:1:6 or 6:3:1) were normalized by the peak intensities of 1:1:1 mixture of three antibodies. Overall, two different methods treated by either PNGase or Fabricator were comparable in estimating the mixture ratios; however, the accuracy and precision data from the Fabricator group were slightly better than PNGase group possibly due to the generation of smaller fragments by Fabricator.

Monoclonal Antibody를 이용한 Streptococcus mutans 검출 방법의 임상적 적용에 관한 연구

홍희정,김종수,김용기 大韓小兒齒科學會 2009 大韓小兒齒科學會誌 Vol.36 No.4

Monoclonal antibody를 이용한 Saliva-check Mutans키트의 타액 Streptococcus mutans검출방법으로서의 활용도와 임상적 우식지수 및 기존의 세균배양방법과의 상관관계를 알아보기 위해 2008년 2월에서 5월 중 평촌키즈웰치과에 내원한 만 2세에서 만 8세 사이의 92명의 아동을 대상으로 Streptococcus mutans 검출검사를 실시하였으며 또한 우식에 영향을 미치는 다른 요인인 치면세균막 pH와 타액완충능력검사를 실시하여 다음과 같은 결론을 얻었다. 1. Saliva-check Mutans 검사결과 양성을 나타낸 아동은 27명으로 29.3%이었고, 음성을 나타낸 아동은 65명으로 70.65%이었다. 우식경험유치면률은 음성 아동 13.89%, 양성 아동 25.23%으로 나타났다. 2. Monoclonal antibody를 이용한 검사방법 인 Saliva- Mutans와 기존의 세균 배양방법인 -SM은 각각 상이한 검사방법을 이용함에도 불구하고 검사결과 높은 상관관계를 보였다(p<0.01). 3. Saliva-check Mutans검사와 치면세균막 pH검사와는 역상관관계를 나타내었으나(p<0.01),타액완충능 검사와는 상관관계가 나타나지 않았다(p>0.05). 이상의 결과로 보았을 때 Monoclonal antibody를 이용한 검사방법인 Saliva-check Mutans는 구강 내 Streptococcus mutans를 측정하는 방법으로 적당하며 또한 검사에 필요한 시간을 대폭 줄일 수 있고 사용방법도 매우 간편하게 개발되어 환자들에게 적용함에 있어 효과적이라고 사료되었다. The purpose of this study was to evaluate the clinical effectiveness of new streptococcus detection system which used monoclonal antibody against Streptococcus mutans. 92 children aged between 2 and 8 were involved in this experiment and their saliva samples were collected for testing. Streptococcus mutans were measured by both monoclonal antibody-based detecting system (Saliva-check Mutans) and dip slide detecting system(Dentocult-SM). The results showed that Saliva-check Mutans levels had a significant correlation with dfs rate of subjects and the two test kits, Saliva-check Mutans and Dentocult-SM were shown to have a good correlation although they were based on different mechanism.

인체 S100A2 단백질에 특이적인 단일클론 항체

김재화,윤선영,김주헌,주종혁,김진숙,이영희,염영일,최용경,최인성,Kim, Jae Wha,Yoon, Sun Young,Kim, Joo Heon,Joo, Jong-Hyuck,Kim, Jin Sook,Lee, Younghee,Yeom, Young Il,Choe, Yong-Kyung,Choe, In Seong 대한면역학회 2003 Immune Network Vol.3 No.1

Background: The S100A2 gene, also known as S100L or CaN19, encodes a protein comprised of 99-amino acids, is a member of the calcium-binding proteins of EF-hand family. According to a recent study, this gene was over-expressed in several early and malignant carcinomas compared to normal tissues. To elucidate the role of S100A2 protein in the process during carcinogenesis, production of monoclonal antibody specific to the protein is essential. Methods: First, cDNA sequence coding for ORF region of human S100A2 gene was amplified and cloned into an expression vector to produce GST fusion protein. Recombinant S100A2 protein and subsequently, monoclonal antibody to the protein were produced. The specificity of anti-S100A2 monoclonal antibody was confirmed by immunoblot analysis of cross reactivity to other recombinant proteins of S100A family (GST-S100A1, GST-S100A4 and GST-S100A6). To confirm the relation of S100A2 to cervical carcinogenesis, S100A2 protein in early cervical carcinoma tissue was immunostained using the monoclonal antibody. Results: GST-S100A2 recombinant protein was purified by affinity chromatography and then fusion protein was cleaved and S100A2 protein was isolated. The monoclonal antibody (KK0723; Korean patent pending #2001-30294) to the protein was produced and the antibody did not react with other members of EF-hand family proteins such as S100A1, S100A4 and S100A6. Conclusion: These data suggest that anti-S100A2 monoclonal antibody produced in this study can be very useful for the early detection of cervical carcinoma and elucidation of mechanism during the early cervical carcinogenesis.

Encephalomyocarditis Virus 표면항원의 단일항체 생산세포주의 크론과 이의 면역학 및 생화학적 연구(Ⅱ)

양종대,박종수,이영탁,김화영,김영래,이인수,조영준,박재윤,차종희,윤지원,고광삼 朝鮮大學校 附設 醫學硏究所 1987 The Medical Journal of Chosun University Vol.12 No.1

To see whether there is any differencies in RNA dependent DNA polymerase activities between monoclonal antibody-producing hydridoma cells and non-producing hybridoma cells, Balb/c female mice were immunized with the purified viral surface protein of D-variant of encephalomyocarditis virus and then fused with myeloma cells (NR-1). After cloring, monoclonal antibody-producing hybridoma cell lines were separated from non-producing hydridoma cell lines. RNA-dependent DNA polymerase activities were measured in the supernatant of monoclonal antibody-producing hybridoma clones and non-producing hybr idoma clones, and myeloma cells as control, Monoclonal antibody-producing hybridoma cells showed statistically significant higher activity as compar compare to that of nonproducing hybridoma cells. To find whether RNA-dependent DNA polymerase releasing cells aware secreting or budding C-type virus particles, those cells were examined with electron microscope. The hybridoma cell which secrete large amount of RNA-dependent DNA polymerase shows significant number of extracellular C-type virus particles. In constrast, non-producing hydridoma cells contains a lot of intracellular C-type virus particles. It is concluded that monoclonal antibody-producing hydridoma cells released particles. It is concluded that monoclonal antibody-producing hydridoma cells released significant amount of RNA-dependent DNA polymerase land extracellular C-type virus particles, while non-producing hydridoma cells showed less release of RNA-dependent DNA polymerase and contains intracellular C-type virus particles.

Studies on the development of enzyme linked immuno-sorbent assay (ELISA) for hepatitis B surface antigen (HBsAg) by monoclonal antibodies of different affinity constants

Kim, Gye-Won,Hong, Sung-Youl,Shin, Soon-Cheon,Lee, Sung-Hee,Kim, Won-Bae The Pharmaceutical Society of Korea 1987 Archives of Pharmacal Research Vol.10 No.1

Mouse monocolonal antibodies to Hepatitis B surface antien (HBsAg) were prepared and their functional capabilities tested by the method of solid phase enzyme linked immuno sorbent assay (ELISA). HBsAg binding studies inicated that one monoclonal antibody 6E-1-1 bound more HBsAg at a faster rate than the other monoclonal antibodies. Also, for the binding inhibition studies with the selected monoclonal antibody 6E-1-1, one monoclonal antibody 8D-3-6 didn't exhibit binding inhibition for HBsAg. Then, a simultaneous ELISA method was developed for the immunodiagnosis of HBsAg. Different combinations of two monoclonal antibodies as solid phase and horseradish peroxidase (HRPO) labeled phase were studied. The combination of monoclonal antibody of higher affinity constant (6E-1-1) immobilized in a solid phase and monoclonal antibody of lower affinity constant (8D-3-6) as a HRPO laeled phase was more sensitive when two monoclonal antibodies of different affinity constants for HBsAg were prepared.