• Biotechnology and Bioprocess Engineering:BBE
• /
• 제11권2호
• /
• pp.173-177
• /
• 2006

In this study, a novel strategy was developed for the highly selective immobilization of proteins, using the polyhydroxyalkanoate (PHA) depolymerase substrate binding domain (SBD) as an active binding domain. In order to determine the appropriacy of this method for immunodiagnostic assays, the single-chain antibody (ScFv) against the hepatitis B virus (HBV) preS2 surface protein and the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVe) were fused to the SBD, then directly immobilized on PH A-coated slides via microspotting. The fluorescence-labeled HBV antigen and the antibody against SCVe were then utilized to examine specific interactions on the PHA-coated surfaces. Fluorescence signals were detected only at the spotted positions, thereby indicating a high degree of affinity and selectivity for their corresponding antigens/antibodies. Furthermore, we detected small amounts of ScFv-SBD (2.7 ng/mL) and SCVe-SBD fusion proteins (0.6ng/mL). Therefore, this microarray platform technology, using PHA and SBD, appears generally appropriate for immunodiagnosis, with no special requirements with regard to synthetic or chemical modification of the biomolecules or the solid surface.

• IMMUNE NETWORK
• /
• 제2권1호
• /
• pp.41-48
• /
• 2002

Background: Viral antigens presented on the cell surface in association with MHC class I molecules are recognized by CD8+ T cells. MHC restricted peptides are important in eliciting cellular immune responses. As peptide antigens have a weak immunigenicity, pH-sensitive liposomes were used for peptide delivery to induce effective cytotoxic T lymphocyte (CTL) responses. In the previous study, as the HBx peptides could induce specific CTLs in vitro, we tested whether the HLA-A2/$K^b$ transgenic mice that were immunized by HBx-derived peptides could be protected from a viral challenge. Methods: HBx-peptides encapsulated by pH-sensitive liposomes were prepared. $A2K^b$ transgenic mice were immunized i.m. on days one and seven with the indicated concentrations of liposome-encapsulated peptides. Three weeks later, mice were infected with $1{\times}10^7pfu$/head of recombinant vaccinia virus (rVV)-HBx via i.p. administration. The ovaries were extracted from the mice, and the presence of rVV-HBx in the ovaries was analyzed using human TK-143B cells. IFN-${\gamma}$ secretion by these cells was directly assessed using a peptide-pulsed target cell stimulation assay with either peptide-pulsed antigen presenting cells (APCs), concanavalin A ($2{\mu}g/ml$), or a vehicle. To generate peptide-specific CTLs, splenocytes obtained from the immunized mice were stimulated with $20{\mu}g/ml$ of each peptide and restimulated with peptide-pulsed APC four times. The cytotoxic activity of the CTLs was assessed by standard $^{51}Cr$-release assay and intracellular IFN-${\gamma}$ assay. Results: Immunization of these peptides as a mixture in pH-sensitive liposomes to transgenic mice induced a good protective effect from a viral challenge by inducing the peptide-specific CD8+ T cells. Mice immunized with $50{\mu}g/head$ were much better protected against viral challenge compared to those immunized with $5{\mu}g$/head, whereas the mice immunized with empty liposomes were not protected at all. After in vitro CTL culture by peptide stimulation, however, specific cytotoxicity was much higher in the CTLs from mice immunized with $5{\mu}g/head$ than $50{\mu}g/head$ group. Increase of the number of cells that intracellular IFN-${\gamma}$ secreting cell among CD8+ T cells showed similar result. Conclusion: Mice immunized with XEPs within pH-sensitive liposome were protected against viral challenge. The protective effect depended on the amount of antigen used during immunization. XEP-3-specific CTLs could be induced by peptide stimulation in vitro from splenocytes obtained from immunized mice. The cytotoxic effect of CTLs was measured by $^{51}Cr$-release assay and the percentage of accumulated intracellular IFN-${\gamma}$ secreting cells after in vitro restimulation was measured by flow cytometric analysis. The result of $^{51}Cr$-release cytotoxicity test was well correlated with that of the flow cytometric analysis. Viral protection was effective in immunized group of $50{\mu}g/head$, while in the in vitro restimulation, it showed more spectific response in $5{\mu}g$/head group.

• Journal of Yeungnam Medical Science
• /
• 제14권2호
• /
• pp.359-369
• /
• 1997

저자들은 1993년 3월에서 1997년 9월까지 영남대학교 의과대학 부속병원 임상병리과에 면역학적 표지자 검사가 의뢰된 153명의 백혈병 환자의 결과를 분석하였다. 여기에는 AML 61례, ALL 46례, FAB 아형이 확정되지 않았던 12례 등이 포함되어 있었다. EDTA 항응고처리한 골수나 말초혈액에서 단핵세포층을 분리한후 일련의 단클론항체와 FITC 표지 이차항체를 이용하여 면역형광염색법을 실시한 후 형광현미경으로 관찰하였다. AML 61례에서의 비전형적인 표지자 표현 양상을 보면 CD7 32.8%, CD10 14.8%, CD5 13.1%, CD2 6.6%, CD4 4.9%, CD19 1.6%의 양성률을 보였고, TdT 양성은 3.9%였다. 혼성 백혈병으로 최종 진단을 내린 경우는 13.1%로 그 중 단구성 계열이 대부분(6/8)이었다. ALL 46례에서의 면역학적 검사에 따른 분류에 따르면 CALLA(+) B precursor형이 65.2%를 차지하였고, CALLA(-) B precursor형이 10.9%, T 세포형 8.7%, B 세포형 2.2%, 혼성림프구형(B&T) 4.3%, 미분화성 백혈병 2.2%였고, 혼성 백혈병으로 진단된 경우는 6.5%였다. 골수성 표지자 양성인 ALL은 CD13이나 CD33 양성인 례가 각각 2.2%였다. 급성 백혈병에서 골수형태와 세포화학검사로 FAB 아형을 확정하지 못한 12례에서 혼성 백혈병이 5례(41.7%)였고, 림프구 계열이 7례(58.3%)였으며 그 중 B 세포계열이 3례, T 세포계열이 3례, 혼성 림프구형(B&T)이 1례로 나타났다. 결론적으로는, 면역학적 표지지 검사로 더 정확하게 AML과 ALL을 감별할 수 있었으며, M0, M7 아형진단에도 도움이 되며, 형태학적, 세포화학적검사로 정확히 진단할 수 없었던 급성 백혈병의 분류에 도움이 되었다. 따라서 급성 백혈병의 진단을 위하여 FAB 분류 뿐 만 아니라 면역학적 표지자 검사를 함께 시행한다면 보다 정확한 진단에 도움을 줄 수 있을 것으로 생각된다.

• 미생물학회지
• /
• 제54권4호
• /
• pp.330-342
• /
• 2018

Streptavidin (STR)과 Biotin system은 Biotin의 Streptavidin에 대한 높은 비공유 친화력(non-covalent affinity; $K_D=10^{-14}M$)과 4 Biotin 결합부위를 갖는 Streptavidin의 tetramer 구조로 인해 복수의 항원결합부위 및 복수의 항원특이성을 갖는 항체를 제조할 수 있기 때문에 가장 활발하게 연구되고 있다. 이 system을 활용하기 위해 우리는 Streptomyces avidinii 염색체 DNA로부터 PCR을 통해 Streptavidin (STR) 유전자를 증폭하고 이를 TRAIL (tumor necrosis factor ${\alpha}$ related apoptosis induced ligand) receptor인 death receptor 4 (DR4)에 특이적으로 결합하는 hAY4 single-chain Fv 항체유전자에 융합시켰다. 대장균에서 발현시킨 STR에 융합된 hAY4 ScFv (hAY4-STR) 항체는 가열시킨 SDS-PAGE에서 43 kDa monomer를 나타내었다. 그러나 가열하지 않은 SDS-PAGE와 Size-exclusion chromatography에서는 tetramer인 172 kDa을 나타내었는데 이는 hAY4 ScFv-STR 항체가 STR의 자연적인 비공유결합에 의해 유도된 tetramer를 형성하고 있음을 나타내고 있다. 본 융합 단백질은 Ouchterlony assay와 ELISA에서 보여주는 것처럼 자연 Streptavidin과 유사한 Biotin 결합력을 유지하고 있었다. ELISA와 Westernblot을 이용하여 정제된 hAY4-STR 융합항체의 DR4 항원결합력 또한 확인하였다. 게다가 표면 플라즈몬 공명(surface plasmon resonance) 분석에서 hAY4 ScFv-STR tetramer는 tetramerization에 의해 hAY4 ScFv monomer보다 60배 더 높은 항원결합력을 나타내었다. 요약하면 hAY4 ScFv-STR 융합단백질은 E. coli에서 soluble tetramer로 성공적으로 발현 및 정제되었으며 Biotin과 DR4 항원에 동시에 결합함을 보여 주었다. 이는 bifunctional and tetrameric ScFv 항체를 제조 할 수 있음을 제시해 주고 있다.

• Animal cells and systems
• /
• 제3권3호
• /
• pp.337-341
• /
• 1999

Hepatitis C virus (HCV) is a positive strand RNA virus of the Flaviviridae family and the major cause of post-transfusion non-A, non-B hepatitis. Vaccine development for HCV is essential but has been slowed by poor understanding of the type of immunity that naturally terminates HCV infection. The DNA-based immunization technique offers the potential advantage of including cellular immune responses against conserved internal proteins of a virus, as well as the generation of antibodies to viral surface proteins. Here, we demonstrate that cell lines expressing the HCV core and/or NS3 proteins can induce a specific CTL response in mice, and these results suggest a possibility that the HCV core and NS3 DNA can be used to induce CTL activity against the antigen in mice and can be further developed as a therapeutic and preventive DNA vaccine.

• Parasites, Hosts and Diseases
• /
• 제38권2호
• /
• pp.107-110
• /
• 2000

We investigated the role of recombinant Toxoplasma gondii heat shock protein (rT.g.HSP) 70-full length, rT.g. HSP70-NH2-terminal region, or rT.g. HSP70-carboxy-terminal region in prophylactic immunity in C57BL/6 mice perorally infected with Fukaya cysts of T. gondii. At 3, 4, 5, and 6 weeks after infection, the number of T gondii in the brain tissue of each mouse was measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene. Immunization with rT.g.HSP70-full length or rT.g.HSP70-carboxy-terminal region increased the number of T. gondii in the brain tissue after T. gondii infection, whereas immunization with rT.g.HSP70-NHa-terminal region did not. These results suggest that T.g. HSP70-carboxy-terminal region as well as T.g.HSP70-full length may induce deleterious effects on the protective immunity of mice infected with a cyst-forming T. gondii strain, Fukaya.

• IMMUNE NETWORK
• /
• 제9권5호
• /
• pp.203-207
• /
• 2009

Background: The FimA of Porphyromonas gingivalis is a crucial pathogenic component of the bacteria and has been implicated as a target for vaccine development against the periodontal diseases. Methods: In this study, the purified fimbriae (FimA subunit polymers) protein was used for immunization in their native form and B hybridoma clones producing antibodies specific to FimA were established. Results: The monoclonal antibodies prepared from selected two clones, designated #123 (IgG2b/ kappa) and #265 (IgG1/kappa), displayed different patterns of binding activity against the cognate antigen. Both antibodies reacted with conformational epitopes expressed by partially dissociated oligomers, but not with monomer as elucidated by Western blot analysis. Ascites fluid containing the monoclonal antibodies showed the inhibitory activity against P. gingivalis to saliva-coated hydroxyapatite beads, an in vitro model for the pellicle-coated tooth surface. Conclusion: These results suggest that the monoclonal antibodies could be used as vaccine material against the periodontal diseases through passive immunization.

• BMB Reports
• /
• 제34권2호
• /
• pp.156-165
• /
• 2001

Leptin is a circulating non-glycosylated protein that is mainly produced in adipocytes. Leptin acts in the brain to regulate food intake and energy expenditure. Previously we reported our success in the isolation of a partial cDNA of the long form of the leptin receptor, OB-Rb, from rat spleen, and showed that leptin might also play a role in peripheral immune organs. In the present study, for the first time, the complete coding region of OB-Rb cDNA was cloned from rat splenocytes, and its nucleotide sequence was determined. The cDNA was then further expressed in E. coli and mammalian cells, thereby confirming the functional integrity of this receptor. Prokaryotically overexpressed OB-R protein was then used as an immunizing antigen in BALE/c mice to produce leptin receptor-specific antibodies. By using them, we confirmed the cell surface expression of OB-Rb in transfected CHO cells. It is our belief that the reagents, as produced in this study, will be of great use in further studies of the biological role of rat leptin.

• 대한기계학회논문집A
• /
• 제28권9호
• /
• pp.1429-1434
• /
• 2004

Immunoassay is one of the important analytical methods for clinical diagnoses and biochemical studies, but needs a long time, troublesome procedures and expensive reagents. In this study, therefore, we propose the micro filter chip with microbeads for immunoassay, which has pillar structures. The advantage of the proposed micro filter chip is to use simple fabrication process and cheap materials. The mold was made by the photolithography technique with Si wafer and negative photoresist SU-8. The replica was made of PDMS, bonded on the pyrex glass. The micro filter chip consists of inlet channel, filter chamber and outlet channel. HBV (Hepatitius B virus) monoclonal antibody (Ag1) labeled with biotin were immobilized onto streptavidin coated beads of 30∼50 $\mu$m size. Fluorescein isothiocyanate (FITC)-labeled HBV monoclonal antibody (Ag8) was used to detect HBsAg (Hebatitis B virus surface Antigen), and fluorescence intensity was monitored by epi-fluorescence microscope. In this study, the immune response of less than 30 min was obtained with with the use of 100 $m\ell$ of sample.

• 대한의용생체공학회:의공학회지
• /
• 제34권3호
• /
• pp.123-128
• /
• 2013

The 'Dielectrophoretic Tweezers(DEP Tweezers)' can be used as a facile, economical toolkit for quantitative measurement of chemical and biological binding forces related to many biological interactions within a microfluidic device. Our experimental setup can probe the interaction between a single receptor molecule and its specific ligand. Immunoglobulin G(IgG) functionalized on polystyrene microspheres has been used to detect individual surface linked Staphylococcus protein A(SpA) molecules and to characterize the strength of the noncovalent IgG-SpA bond. It was measured and compared with the existing measurements. Measured single binding force of between Goat, Rabbit IgG and SpA were $17{\pm}7pN$, $74{\pm}16pN$. This work can be used to investigate several different ligand-receptor interactions and antigen-antibody interactions.