• Title, Summary, Keyword: monoclonal antibody

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Development and Immunochemical Properties of Two Monoclonal Antibodies Specific to Human Chorionic Gonadotropin

  • Kim, You-Hee;Koh, Kwan-Sam
    • BMB Reports
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    • v.32 no.5
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    • pp.474-479
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    • 1999
  • Using a hybridoma technique, spleen cells of Balb/c mice immunized with human chorionic gonadotropin (hCG) were fused with NS-1 mouse myeloma cells. Two hybrid cell lines, clones KS-8 and KS-19, secreting monoclonal antibodies to hCG, were isolated. KS-8 and KS-19 belong to the immunoglobulin $G_1$ subclass. With the aid of a double-antibody radioimmunoassay, it was established that the KS-8 monoclonal antibody recognizes an immunodeterminant of the $\beta$-subunit of hCG, whereas the KS-19 monoclonal antibody recognizes an epitope present on the $\alpha$-subunit of hCG. The KS-8 monoclonal antibody specifically reacts with human chorionic gonadotropin and shows cross-reactivity of less than 0.3% to other related human glycoprotein hormones. On the other hand, using a hemagglutination test based on antibody-induced agglutination of sheep red blood cells coated with hCG, It was shown that only the KS-19 monoclonal antibody was capable of inducing a positive reaction, although both monoclonal antibodies had similar binding capacity to the coated cells. The results from the dual screening procedures demonstrate that KS-8 and KS-19 monoclonal antibodies show high sensitivity in two different assays, and are hence useful for the qualitative and quantitative determination of hCG by both radioimmunoassay and hemagglutination inhibition tests.

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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
    • Archives of Pharmacal Research
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    • v.10 no.1
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    • pp.18-24
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    • 1987
  • 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.

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Application of monoclonal antibody to develop diagnostic techniques for infectious bovine rhinotracheitis virus. II. Diagnosis of infectious bovine rhinotracheitis by using monoclonal antibody (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))

  • Jun, Moo-hyung;Kim, Duck-hwan;An, Soo-hwan;Lee, Jung-bok;Min, Won-gi
    • Korean Journal of Veterinary Research
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    • v.29 no.1
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    • pp.27-35
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    • 1989
  • 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.

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Production and characterization of a monoclonal anti-glutathione-S-transferase(GST) antibody

  • You, Je-Kyung;Shin, Chan-Young;Park, Kyu-Hwan;Ko, Kwang-Ho
    • Proceedings of the Korean Society of Applied Pharmacology
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    • pp.93-93
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    • 1997
  • Analysis of protein is often frustrated by the inability to isolate large amounts of purified protein from a native source. To overcome this problem, fusion protein expression systems such as pGEX system have been widely used. Using pGEX system, the desired protein could be easily obtained in a large amount in E. coli, and then the fusion protein could be used for the study of the function of the given protein. To analyze and purify the GST fusion protein, anti-GST antibody could be used as one of the system of choice. However, the production and characterization of monoclonal anti-GST antibody has not been studied extensively yet. To produce monoclonal anti-GST antibody, GST was purified from E. coli transformed with pGEX-cs, one of the pGEX system and was used as an antigen. The monoclonal antibody was produced by fusion of the immunized spleen cells with SP2-0 myeloma cells. The antibody was characterized by ELISA, western blotting, etc. The monoclonal antibody produced in this study (mAb-GSTA) showed strong and specific immunoreactivity against not only GST but also GST-fusion proteins. Also, mAb-GSTA was successfully used for the immunoaffinity purification of the GST ${\beta}$-Rc.-third intracellular-loop fusion protein. The results of the present study suggest that mAb-GSTA may be used for the identification and purification of GST fusion proteins.

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Characterization of the Monoclonal Antibody Specific to Human S100A6 Protein (인체 S100A6 단백질에 특이한 단일클론 항체)

  • Kim, Jae Wha;Yoon, Sun Young;Joo, Joung-Hyuck;Kang, Ho Bum;Lee, Younghee;Choe, Yong-Kyung;Choe, In Seong
    • IMMUNE NETWORK
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    • v.2 no.3
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    • pp.175-181
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    • 2002
  • Background: S100A6 is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity and involved in various cellular processes such as cell division and differentiation. To detect S100A6 protein in patient' samples (ex, blood or tissue), it is essential to produce a monoclonal antibody specific to the protein. Methods: First, cDNA coding for ORF region of human S100A6 gene was amplified and cloned into the expression vector for GST fusion protein. We have produced recombinant S100A6 protein and subsequently, monoclonal antibodies to the protein. The specificity of anti-S100A6 monoclonal antibody was confirmed using recombinant S100A recombinant proteins of other S100A family (GST-S100A1, GST-S100A2 and GST-S100A4) and the cell lysates of several human cell lines. Also, to identify the specific recognition site of the monoclonal antibody, we have performed the immunoblot analysis with serially deleted S100A6 recombinant proteins. Results: GST-S100A6 recombinant protein was induced and purified. And then S100A6 protein excluding GST protein was obtained and monoclonal antibody to the protein was produced. Monoclonal antibody (K02C12-1; patent number, 330311) has no cross-reaction to several other S100 family proteins. It appears that anti-S100A6 monoclonal antibody reacts with the region containing the amino acid sequence from 46 to 61 of S100A6 protein. Conclusion: These data suggest that anti-S100A6 monoclonal antibody produced can be very useful in development of diagnostic system for S100A6 protein.

Protective Effects of a Monoclonal Antibody to a Mannose-Binding Protein of Acanthamoeba culbertsoni

  • Park, A-Young;Kang, A-Young;Jung, Suk-Yul
    • Biomedical Science Letters
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    • v.24 no.4
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    • pp.435-438
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    • 2018
  • Acanthamoeba culbertsoni is the causative agent of granulomatous amoebic encephalitis (GAE), a condition that predominantly occurs in immunocompromised individuals and which is typically fatal. A mannose-binding protein (MBP) among lectins was shown to have strong A. castellanii pathogenic potential when correlated with major virulence proteins. In this study, protective effects were analyzed using the monoclonal antibody to A. culbertsoni MBP by quantification and were also compared with other free-living amoebae. For the amoebial cytotoxicity to the target cell, amoeba trophozoites were incubated with Chinese hamster ovary (CHO) cells. For the protective effects of antibodies, amoebae were pre-incubated with them for 4 h and then added to the target cells. After 24 h, the supernatants were collected and examined for host cell cytotoxicity by measuring lactate dehydrogenase (LDH) release. The cytotoxicity of A. culbertsoni to the CHO cells showed about 87.4%. When the monoclonal antibody was pre-incubated with A. culbertsoni, the amoebial cytotoxicity was remarkably decreased as shown at LDH release (1.858 absorbance), which was represented with about 49.9%. Taken together, it suggested that the monoclonal antibody against MBP be important to inhibit the cytotoxicity of A. culbertsoni trophozoites to the target cell. The antibody will be applied into an in vivo functional analysis, which would help to develop therapeutics.

Characteristics and application of monoclonal antibody to progesterone II. Development of progesterone enzyme-linked immunosorbent assay(ELISA) (Progesterone의 단크론성 항체에 관한 특성 및 활용에 관한 연구 II. ELISA 기법의 개발)

  • Kang, Chung-boo;Kim, Jong-shu
    • Korean Journal of Veterinary Research
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    • v.31 no.4
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    • pp.403-409
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    • 1991
  • This experiment was carried out to develop a sensitive, rapid, solid-phase microtitre plate assay of progesterone using the monoclonal antibody to this hormone. Monoclonal antibody to progesterone was much higher titre and binding affinity about 10 times than conventional polyclonal antibody to progesterone. Dot-blot analysis of monoclonal antibody revealed a single precipitation band when reacted with anti-mouse IgM and anti-mouse K. A competitive reaction was used with a reaction time of 2 hours. The standard dose-response curve was linear through 1,000pg/well. This ELISA system approach is applicable to evaluation for the rapid assessment of luteal function and reproductive status in both clinical and research in a wide variety of species.

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Study on Development of Enzyme-Linked Immunosorbent Assay for the Screening of Chloramphenicol Residues (잔류 Chloramphenicol 검사용 효소 면역측정법의 개발에 관한 연구)

  • 윤동호;이문한
    • Journal of Food Hygiene and Safety
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    • v.8 no.4
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    • pp.205-214
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    • 1993
  • The monoclonal antibody to chloramphenicol(CAP) was produced to develop an enzyme-linked immunosorbent assay(ELISA) for residual CAP. An immunogen(CAP-BSA) was prepared by immunogen, antibody titer was measured by indirect ELISA. Spleen cells form the immunized mouse were fused with SP2/OAg14 myeloma cells. Among hybridomas selected in HAT media, 6 clones shown high antibody titer to CAP were subjected to cloning by limit dilution, and all of the monoclonal antibodies(MCA1, 2, 3, 4, 5, 7 and 9) produced by each clone were identified as IgG1 by ELISA isotyping analysis. Competitive ELISA condition was established by using the purified monoclonal antibody MCA1 as primary antibody and CAP-HSA conjugate as coating antigen. Standard curve of CAP(n=28) showed that the lowest detection limit of CAP is 20ng/ml level. The cross-reactivities of the 6 monoclonal antibodies showed that CAP sodium succinate. CAP base, P-nitrophenol, and p-nitrobenzyl alcohol were 89∼178, 0.050∼2.237, 0.056∼0.794 and 0.013∼7.939%, respectively. No cross-reactivities were observed with phenylalanine, tyrosine, glutamine, thiamphenicol, neomycin, streptomycin, gentamicin, sulfamethazine, sulfathiazole, chlortetracycline and p-aminobenzoic acid.

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Detection of Fish Virus by Using Immunomagnetic Separation and Polymerase Chain Reaction (IMS-PCR)

  • KIM Soo Jin;OH Hae Keun;CHOI Tae-Jin
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.30 no.6
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    • pp.948-955
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    • 1997
  • Immunomagnetic separation of virus coupled with .reverse transcription-polymerase chain reaction (IMS-PCR) was performed with infectious hematopoietic necrosis virus (IHNV). A DNA fragment of expected size was synthesized in the RT-PCR with total RNA extracted from IHNV inoculated CHSE-214. In a SDS-PAGE analysis, a protein band of over 70kDa was detected from non-infected cells and cells inoculated with IHNV and infectious pancreatic necrosis virus (IPNV). This protein was detected in the Western blot analysis probably because of non-specific reaction to monoclonal antibody against IHNV nucleocapsid protein. In the immunomagnetic separation, magnetic beads coated with monoclonal antibody against the IHNV nucleocapsid protein was incubated with supernatant from IHNV inoculated CHSE-214 cells. During this process, the non-specifically reacting protein could be removed by washing the magnetic bead with PBS in the presence of an external magnetic field, and viral proteins were detected from the remaining, cleaned magnetic beads. It was necessary to extract viral RNA from the captured virus particles before RT-PCR, and no DNA product was detected when the captured virus was only heated 5 min at $95^{\circ}C$. A PCR-product of expected size was synthesized from IMS-PCR with magnetic beads double coated either by goat anti-mouse IgG antibody -monoclonal antibody or streptavidin - biotin conjugated monoclonal antibody.

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Monoclonal antibody의 대량 생산을 위한 hybridoma cell의 생존능 증가에 관한 연구

  • Ha, Seong-Jin;Im, Seon-Ha;Lee, Jong-Won;Jo, Mu-Hwan
    • 한국생물공학회:학술대회논문집
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    • pp.561-562
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    • 2003
  • Hybridoma cell is very important in point of producing monoclonal antibody(Mab). Producing large quantity of Mab is economically valuable. On this experiment, we used one of hybridoma cell line, 5F12 AD3, and treated various antibiotics such as genetitin(G418), ciprofloxacin and minocycline to improve cell viability and we expect that improving cell viability brings higher concentrations of Mab. The optimum concentration of each antibiotics for improving cell viability were 10ug/ml for G418, 1ug/ml or 10ug/ml for ciprofloxacin and 1ug/ml for minocycline.

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