• IMMUNE NETWORK
• /
• 제8권3호
• /
• pp.75-81
• /
• 2008

Background: Platelets take part in repairing the lesions of endothelial damage. To understand the molecular mechanism of this process, we tested the hypothesis that CD154 expressed on activated platelets stimulates proliferation of human endothelial cells. Methods: The expression levels of CD154 and CD40 on platelets and endothelial cells, respectively, were measured by flow cytometry and confocal microscopy. Function-blocking monoclonal antibody against CD154 was developed after immunization with CD154-transfected L cells. Results: An anti-CD40 agonist antibody and soluble CD154 both induced significant proliferation of endothelial cells. In addition, a function-blocking anti-CD154 antibody inhibited the platelet-induced proliferation of endothelial cells, indicating that the CD154-CD40 pathway is involved in these cellular interactions. An anti-VEGF antibody failed to inhibit the proliferation. This, in addition to the fact that very small amounts of VEGF are released from platelets or endothelial cells, suggests that VEGF does not play an important role in the platelet-stimulated proliferation of endothelial cells. Conclusion: Our results indicate that platelets induce proliferation of endothelial cells by CD154-CD40 interactions independently of VEGF.

• 약학회지
• /
• 제37권4호
• /
• pp.420-426
• /
• 1993

To effectively purify of IL-1 inhibitor from human febrile urine, we have established monoclonal antibody that reacts with human recombinant interleukin l$\beta$(IL-1$\beta$). The antibody, designated ON-1, was highly specific to IL-1$\beta$ and no cross-reaction with other cytokines(IL-l$\alpha$ and IL-4) was observed. As the results of ELISA inhibition assay and Western blotting method, it was further identified that ON-1 had high binding specificity with IL-1$\beta$. IL-1 receptor binding material from febrile patient urine was effectively purified with affinity column chromatography which conjugated with ON-1. This urinary material inhibited the thymocyte proliferation in a dosedependent manner. IL-l$\beta$ induced thymocyte proliferation activity was inhibited to 67.3% at 6 $\mu\textrm{g}$ of the purified urinary material. The result may suggest that this urinary material the purified urinary material. The result may suggest that this urinary material will have antagonic effect on IL-1 action mechanism and act IL-l$\beta$ inhibitor.

• 센서학회지
• /
• 제17권5호
• /
• pp.380-386
• /
• 2008

Quartz crystal microbalance immunosensor has a capacity to perform a label-free and real time detection of a trace amount of analyte through the specific interaction between antibody and antigen. However, immobilization of antibody molecules on the sensor surface is a troublesome procedure for researchers who are not experienced in chemistry. Protein G has a specific affinity to antibody and would serve as a capturing agent for antibody when immobilized on the sensor surface. In this work, we prepared a protein G sensor chip by immobilizing protein G on the surface of quartz crystal microbalance and examined its capability to detect human haptoglobin or human transferrin with unpurified corresponding antiserum. Specific and dose dependent response was observed when the protein G chip was used for detection of antigens after saturated with antiserum. We also verified several advantageous aspects of the protein G chip such as improved flexibility and sensitivity.

• 대한핵의학회지
• /
• 제11권1호
• /
• pp.9-15
• /
• 1977

난포 자극 홀몬(hFSH)을 클로라민 티를 사용하여 방사성 요오드로 표지하였으며 평균표지수율은 대략 65%이었다. 표지홀몬을 방사면역측정용으로 사용하기 위하여 전분젤 전기영동과 젤 여과법으로 분리정제하고 그 분리정제효과를 분석한 결과 젤 여과법이 분리시간, 간편성, 항체와의 결합력등으로 보아 우수함을 알 수 있었다. 한편 유리 표지홀몬과 항체와 결합한 표지홀몬의 비율을 결정하기 위하여 이중 항체법을 크로마토 전기영동법과 비교하여 본 결과 이중항체법에 의해서만 효과적 비율결정이 가능하였다.

• Journal of Microbiology and Biotechnology
• /
• 제32권12호
• /
• pp.1615-1621
• /
• 2022

Tissue regeneration is the ultimate treatment for many degenerative diseases, however, repair and regeneration of damaged organs or tissues remains a challenge. Previously, we showed that B1 Ab and H3 Ab induce stem cells to differentiate into microglia and brown adipocyte-like cells, while trafficking to the brain and heart, respectively. Here, we present data showing that another selected agonist antibody, P1 antibody, induces the migration of cells to the pancreatic islets and differentiates human stem cells into beta-like cells. Interestingly, our results suggest the purified P1 Ab induces beta-like cells from fresh, human CD34⁺ hematopoietic stem cells and mouse bone marrow. In addition, stem cells with P1 Ab bound to expressed periostin (POSTN), an extracellular matrix protein that regulates tissue remodeling, selectively migrate to mouse pancreatic islets. Thus, these results confirm that our in vivo selection system can be used to identify antibodies from our library which are capable of inducing stem cell differentiation and cell migration to select tissues for the purpose of regenerating and remodeling damaged organ systems.

• IMMUNE NETWORK
• /
• 제4권1호
• /
• pp.7-15
• /
• 2004

Background: The heat shock proteins (HSPs) play an important role in cellular protection mechanisms against physical or chemical stresses. In this study scFv antibodies specific for human HSP70.1 were isolated from a semi-synthetic human scFv library with the ultimate goal of developing anti-HSP70.1 intracellular antibody (intrabody) that may offer an attractive alternative to gene targeting to study the function of the protein in cells. Methods: A semi-synthetic human scFv display library ($5{\times}10^{8}$ size) was constructed using pCANTAB-5E vector and the selection of the library against bacterially expressed recombinant human HSP70.1 was attempted by panning. Results: Three positive clones specific for recombinant HSP70.1 were identified. All three clones used $V_{H}$ subgroup III. On the other hand, $V_{L}$ of two clones belonged to the kappa light chain subgroup I, but the other utilized $V_{k}$ subgroup IV Interestingly, these scFv molecules specifically reacted to the recombinant HSP70.1, yet failed to recognize native HSP70 induced in U937 human monocytic cells by heat treatment. Conclusion: Our results indicated that affinity selection of an scFv phage display library using recombinant antigens produced in E. coli might not guarantee the isolation of scFv antibody molecules specific for a native form of the antigen. Therefore, the source of target antigens needs to be chosen carefully in order to isolate biofunctional antibody molecules.

• Journal of Microbiology and Biotechnology
• /
• 제14권6호
• /
• pp.1267-1274
• /
• 2004

Colostrum contains various kinds of cytokines including TGF-$\beta$ that has potent regulatory effects on cells of the immune system. We compared the levels of TGF-$\beta$1 and TGF-$\beta$2 in bovine and human colostrum. Based on the isoform-specific ELISA, bovine colostrum collected on day 1 post-delivery retained $53.71{\pm}29.55\;ng/ml$ of TGF-$\beta$1 and $40.41{\pm}21.78\;{\mu}g/ml$ of TGF-$\beta$2 (n=4), while in human, $381.45{\pm}158.24\;ng/ml$ of TGF-$\beta$1 and $41.47{\pm}9.63\;ng/ml$ of TGF-$\beta$2 (n=5). Thus, dominant TGF-$\beta$ isoforms were completely opposite between human and bovine colostrum samples. The concentrations of both isoforms declined as lactation proceeded. Biological activities of the colostrum samples were determined using an MV1LU cell line. Consistent with the result from the immunoassay, TGF-$\beta$1 in human and TGF-$\beta$2 in bovine colostrum were responsible for the anti proliferative activity against MV1LU cells. Furthermore, bovine colostrum increased IgA secretion by LPS-stimulated mesenteric lymph node (MLN) cells, and this effect was abrogated by either anti­TGF-$\beta$2 antibody or combined anti-TGF-$\beta$1/$\beta$2 antibody, but not by anti- TGF-$\beta$1 antibody alone. Similarly, TGF-$\beta$2 in bovine colostrum enhanced the Ig germ line (GL) promoter activity, which is the earliest event toward IgA isotype switching. Taken together, these results suggest that TGF-$\beta$ isoforms, differentially expressed in human and bovine colostrum, may promote IgA isotype production in the neonatal intestine.

• 한국안광학회지
• /
• 제12권2호
• /
• pp.79-86
• /
• 2007

본 연구는 mycoplasma가 오염된 배양 각막 상피세포에 anti-FAS and anti-FAS ligand antibody를 노출시킨 후 세포고사 메커니즘을 조사하기 위해 시행하였다. 배양각막 상피세포에 anti-FAS antibody 와 anti-FAS ligand antibody를 2일과 4일 동안 처리하여 주어진 기간 동안 배양하였다. 배양 중인 각막상피세포가 mycoplasma sp.에 의해 오염된 것이 밝혀졌다. mycoplasma removal agent(MRA)가 세포주로부터 세균을 제거하기 위해 이용되었다. MRA를 세포주에 첨가하여 1주일 동안 배양하였다. 그 후 각막상피 세포주는 MRA가 포함되지 않는 배양액에서 수세대 동안 배양되어 커버글라스에 계대배양하여 50-80% 채워졌을 때 MRA 제거여부를 확인하기 위한 검사 키트에 제공된 Hoechst staining을 이용하여 염색하였다. 세포고사 실험은 MRA 제거 전과 제거 후에 시행되었다. mycoplasma가 오염된 배양 각막상피세포에 대한 anti-FAS and anti-FAS ligand antibody의 영향을 알아보기 위해 Hoechst 33342 staining과 Annexin V-FITC and Propidium Iodide Staining을 이용하여 세포고사 유도를 확인하였다. 본 연구는 anti-FAS antibody가 배양각막 상피세포에서 시간과 농도에 비례하는 메커니즘으로 세포고사를 유도한다. mycoplasma가 오염된 세포주는 FAS 유도 세포고사의 민감성을 증가시키는 것으로 나타났다.

• Animal cells and systems
• /
• 제8권2호
• /
• pp.125-133
• /
• 2004

We have extended our previous work that cross-linking CD4 molecules using specific MAb induced antigen nonspecific, MHC unrestricted killing of virally infected target cells by CD$4^+$We have extended our previous work that cross-linking CD$4^+$ molecules using specific MAb induced antigen nonspecific, MHC unrestricted killing of virally infected target cells by CD$4^+$ T cells. The killing activity of antibody activated CD$4^+$T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylamaleimide, a protein kinase C (PKC) inhibitor. Herbimycin A treated human or bovine peripheral blood CD$4^+$T cells lacked PTK activity and failed to kill virally infected target cells even after cross-linking of CD4 molecules. The CD$4^+$cross-linking failed to induce effector cell proliferation or the transcription of TNF${\beta}$ Upregulation of TNF${\beta}$ was induced by incubating the antibody activated effector cells with BHV-1 infected D17 target cells for 10 h. Anti-TNF${\beta}$ antibody partially abolished (13-44%) the direct effector cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70 to 100% of antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector and target cell ratio. These results support the hypothesis that increased p$56^ICK enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines known to participate in target cell killing were produced. A better understanding of the killing activity displayed by CD$4^+$T lymphocytes following surface receptor cross-linking will provide insight into the mechanisms of cytotoxic activity directed toward virally-infected cells.T cells. The killing activity of antibody activated CD$4^+$T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylamaleimide, a protein kinase C (PKC) inhibitor. Herbimycin A treated human or bovine peripheral blood CD4T cells lacked PTK activity and failed to kill virally infected target cells even after cross-linking of CD4molecules. The CD4 cross-linking failed to induce effector cell proliferation or the transcription of TNF$\beta$. Upregulation of TNF$\beta$ was induced by incubating the antibody activated effector cells with BHV-1 infected D17 target cells for 10 h. Anti-TNF$\beta$ antibody partially abolished (13-44%) the direct effector cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70 to 100% of antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector and target cell ratio. These results support the hypothesis that increased $56^ICK enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines known to participate in target cell killing were produced. A better understanding of the killing activity displayed by CD$4^+$T lymphocytes following surface receptor cross-linking will provide insight into the mechanisms of cytotoxic activity directed toward virally-infected cells.

• 한국가축번식학회지
• /
• 제12권1호
• /
• pp.51-62
• /
• 1988

hCG로 면역화된 생쥐의 비장세포와 골수종양세포(SP 2/0 Ag14)를 융합하여 hCG에 대한 단일클론항체를 생산하는 잡종세포(hybridoma)를 얻었다. 생산된 면역글로부린 type과 titer 그리고 면역분석법 이용시 감도 등을 조사함으로써 그 특성을 조사하였다. 배지나 복수내에 존재하는 항체를 순수분리 정제하기 위하여 gel-filtration, DEAE-ion exchange chromatography, 그리고 affinity chromatography 원리에 의한 방법 등을 사용하여 전기영동(SDS-PAGE)으로 순수도를 조사함으로 상호 비교하였다. 또한 hCG의 정량을 위하여 항체를 plastic tube에 피복시킨 것과 화학발광체로 표지된 항체를 이용한 소위 two-site immunochemiluminometric assay(ICMA)를 개발하여 생산된 항체의 이용가능성을 제시하였다.