• Journal of the Institute of Electronics Engineers of Korea SC
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• v.48 no.4
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• pp.102-107
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• 2011

In this paper, a microfluidic array biochip for simultaneously detecting multiple antigen-antibody bindings was designed and implemented. The biochip has the single channel in which microreaction chambers are serially connected, and the antibody-coated microbeads are packed in each microreaction chamber. In addition, the weir structure was fabricated in the microchannel using the gray-scale photolithography in order to trap the microbeads in the microreaction chamber. Three kinds of antibodies were chosen, and the antibodies were immobilized onto the microbeads by the streptavidin-biotin conjugation. In the experiment, as the fluorescence-labeled antigens were injected into the microchannel, the antigen-antibody bindings were completed in 10 minutes. When the solution with multiple antigens was injected into the microchannel, it was observed that the fluorescence intensity increased in only the corresponding microreaction chambers with few non-specific binding. The microfluidic array biochip implemented in this study provides, even with the consumption of tiny amount of sample and fast reaction time to simultaneously detect multiple immunoreactions.

• Macromolecular Research
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• v.17 no.3
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• pp.174-180
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• 2009

Chemical modification of titanium/titanium oxide (Ti/$TiO_2$) substrates has recently gained a great deal of attention because of the applications of Ti/$TiO_2$-based materials to biomedical areas. The reported modification methods generally involve passive coating of Ti/$TiO_2$ substrates with protein-resistant materials, and poly(ethylene glycol) (PEG) has proven advantageous for bestowing a nonbiofouling property on the surface of Ti/$TiO_2$. However, the wider applications of Ti/$TiO_2$ based materials to biomedical areas will require the introduction of biologically active moieties onto Ti/$TiO_2$, in addition to nonbiofouling property. In this work, we therefore utilized surface-initiated polymerization to coat the Ti/$TiO_2$ substrates with polymers presenting the nonbiofouling PEG moiety and subsequently conjugated biologically active compounds to the PEG-presenting, polymeric films. Specifically, a Ti/$TiO_2$ surface was chemically modified to present an initiator for atom transfer radical polymerization, and poly(ethylene glycol) methacrylate (pEGMA) was polymerized from the surface. After activation of hydroxyl groups of poly(pEGMA) (pPEGMA) with N,N'-disuccinimidyl carbonate, biotin, a model compound, was conjugated to the pPEGMA films. The reactions were confirmed by infrared spectroscopy, X-ray photoelectron spectroscopy, contact angle goniometry, and ellipsometry. The biospecific binding of target proteins was also utilized to generate micropatterns of proteins on the Ti/$TiO_2$ surface.

• Journal of Microbiology and Biotechnology
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• v.31 no.5
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• pp.705-709
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• 2021

Porphyromonas gingivalis (P. gingivalis) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer's disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis. First, we performed a visual membrane immunoassay using 3,3',5,5'-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 10³-10⁵ bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without cross-reactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii. Furthermore, three clinical strains of P. gingivalis, KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis.

• Journal of Korean Neurosurgical Society
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• v.30 no.6
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• pp.683-687
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• 2001

Objective : Angiogenesis, the proliferation of capillary endothelial cells, is a vital component in the development, progression, and metastasis of many human tumors. Vascular endothelial growth factor(VEGF) is an endothelial cell-specific mitogen and induces angiogenesis and vascular permeability. The features of glioblastoma, distinct from low grade astrocytomas, are the presence of necroses and vascular endothelial proliferation. In this study, we investigated VEGF expression in the different grades of astrocytomas and determined whether VEGF expression correlates with development of glioblastoma and progression of astrocytomas. Patients and Methods : Forty seven patients with astrocytic tumors(24 males and 23 females), aged 3 to 65 years, were evaluated. Immunohistochemical staining was carried out using labelled streptavidin biotin method and primary antibody was a antirabbit polyclonal Ab against N-terminus region of VEGF165(Oncogene research product, MA, USA). Immunoreactivity(IR) was classified into no IR(absent or a trace of stain), moderate IR and intense IR by level of staining amount and intensity. Results : Six pilocytic astrocytomas showed 3 no IR and 3 moderate IR, 10 astrocytomas showed 2 no IR, 6 moderate IR and 2 intense IR, 12 anaplastic astrocytomas showed I no IR, 7 moderate IR and 4 intense IR and 19 glioblastomas showed 1 no IR, 11 moderate IR and 7 intense IR. Immunoreactivity was significantly different between low and high grade of tumors but there was no significant difference between anaplastic astrocytomas and glioblastomas. Gemistocytic tumor cells represented the predominent VEGF-immunoreactive cell types, as compared with compactly-arranged small tumor cells. In glioblastomas VEGF IR was observed in both perinecrotic and vital tumor areas. Conclusion : VEGF seems to be a important angiogenic factor in anaplastic astrocytomas and glioblastomas and VEGF expression may contribute to neovascularization of human astrocytomas.

• Journal of Microbiology and Biotechnology
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• v.18 no.9
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• pp.1606-1612
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• 2008

Interleukin (IL)-32 is a recently identified proinflammatory cytokine that is one of the IL-18 inducible genes, and plays an important role in autoimmune and inflammatory diseases. We produced antibodies against IL-32 and studied the expression of IL-32 in human stomach cancer. We detected IL-32 secreted from K-562 cells which were stably transfected with IL-32 and in the sera of stomach cancer patients by a sandwich ELISA using a monoclonal antibody KU32-52 and a polyclonal antibody. In order to optimize a sandwich immunoassay, recombinant IL-32a was added, followed by the addition of a biotinylated KU32-52 into microtiter plate wells precoated with a goat anti-IL-32 antibody. The bound biotinylated KU32-52 was probed with a streptavidin conjugated to HRP. This sandwich ELISA was highly specific and had a minimal detection limit of 80 pg/ml (mean${\pm}$SD of zero calibrator) and measuring up to 3,000 pg/ml. This ELISA showed no cross-reaction with other cytokines such as hIL-1$\alpha$, hIL-1$\beta$, hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-$\alpha$. Intra-assay coefficients of variation were 18.5% to 4.6% (n=10), and inter-assay coefficients were 23% to 9% (n=10). The average IL-32 level in the sera of 16 stomach cancer patients (189 pg/ml) was higher than that of 12 healthy control men (109 pg/ml). Our results indicate that serum IL-32 level can be detected by using an established ELISA, and that this immunoassay and mAb KU32-09 specific for immunohistochemistry can be used in the detection of expressed and secreted IL-32 in stomach cancer patients.

• Journal of Pharmaceutical Investigation
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• v.29 no.2
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• pp.87-92
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• 1999

Drug delivery to the brain may be achieved by producing chimeric peptide, attaching the drug to protein 'vectors' which are transported into the brain from the blood by a receptor-mediated transcytosis through the blood-brain barrier (BBB). Since the BBB expresses high concentrations of transferrin receptor, and it was reported that anti-transferrin receptor mouse monoclonal antibody (OX26) undergoes transcytosis through the BBB, it is logical to assume that a drug delivery system via transferrin receptor-mediated transcytosis is a promising strategy. In the present study, therefore, we tested feasibility of several OX26 based vectors for the brain delivery of a model drug. Avidin-based delivery vectors such as OX26-streptavidin (OX26-SA), OX26-neutralite avidin (OX26-NLA) were chemically synthesized vectors and OX26 immunoglobulin G 3 type $C_{H}3$ fusion avidin $(OX26\;IgG3C_H3-AV)$ was genetically engineered. To improve the efficiency of producing chimeric peptide, we used avidin-biotin technology. Pharmacokinetics of $[^3H]biotin$ bound to OX26-SA, OX26-NLA and $OX26\;IgG3C_H3-AV$ was determined by intravenous injection technique, and their stabilities in plasma were analyzed using HPLC. The brain delivery of $[^3H]biotin$ bound to OX26-SA, OX26-NLA and OX26\;$IgG3C_{H}3-AV$ (expressed as %ID/g brain) was $0.22{\pm}0.01$, $0.18{\pm}0.01$ and $0.25{\pm}0.09$, respectively. The areas under the plasma concentration versus time curve (AUC) for OX26-SA, OX26-NLA, $OX26\;IgG3C_H3-AV$ from time zero to 60 min were $209{\pm}10$, $195{\pm}9$, $134{\pm}29\;%ID\;min/ml$ respectively and their total clearances $(CL_{tot})$ were $1.00{\pm}0.09$, $1.08{\pm}0.07$ and $1.54{\pm}0.29\;ml/min/kg$, espectively. These results showed that these vectors possess preferable pharmaceutical (e.g., resonable stability) and pharmacokinetics (e.g., significant brain uptake and enhanced AUC) for brain delivery. Therefore, these vectors may be broadly useful in the brain delivery of drugs that are not transported into the brain to a significant extent.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.2
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• pp.79-84
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• 2003

We have previously reported that expression of the somatostatin receptor subtypes, sst1-5, is differentially regulated by growth hormone (GH)-releasing hormone (GHRH) and forskolin (FSK), in vitro. GHRH binds to membrane receptors selectively located on pituitary somatotropes, activates adenylyl cyclase (AC) and increases sst1 and sst2 and decreases sst5 mRNA levels, without significantly altering the expression of sst3 and sst4. In contrast FSK directly activates AC in all pituitary cell types and increases sst1 and sst2 mRNA levels and decreases sst3, sst4 and sst5 expression. Two explanations could account for these differential effects: 1) GHRH inhibits sst3 and sst4 expression in somatotropes, but this inhibitory effect is masked by expression of these receptors in unresponsive pituitary cell types, and 2) FSK inhibits sst3 and sst4 expression levels in pituitary cell types other than somatotropes. To differentiate between these two possibilities, somatotropes were sequentially labeled with monkey anti-rat GH antiserum, biotinylated goat anti-human IgG, and streptavidin-PE and subsequently purified by fluorescent-activated cell sorting (FACS). The resultant cell population consisted of 95% somatotropes, as determined by GH immunohistochemistry using a primary GH antiserum different from that used for FACS sorting. Purified somatotropes were cultured for 3 days and treated for 4 h with vehicle, GHRH (10 nM) or FSK ($10{\mu}M$). Total RNA was isolated by column extraction and specific receptor mRNA levels were determined by semi-quantitative multiplex RT-PCR. Under basal conditions, the relative expression levels of the various somatostatin receptor subtypes were sst2＞sst5＞sst3=sst1＞ sst4. GHRH treatment increased sst1 and sst2 mRNA levels and decreased sst3, sst4 and sst5 mRNA levels in purified somatotropes, comparable to the effects of FSK on purified somatotropes and mixed pituitary cell cultures. Taken together, these results demonstrate that GHRH acutely modulates the expression of all somatostatin receptor subtypes within GH-producing cells and its actions are likely mediated by activation of AC.

• Journal of Chest Surgery
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• v.31 no.12
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• pp.1200-1205
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• 1998

Background: Lung cancer formation is a multistage process involving activation of protooncogene and inactivation of tumor suppressor genes. We evaluate the significance of cyclin D1, p53, bcl-2 gene mutations in patients with curatively resected stage IIIA non-small cell lung cancer(NSCLC). Material and Method: One hundred consecutive cases of stage IIIA lung cancers from patients operated on curatvely between 1990 and 1995 for which adequate paraffin blocks and clinical history were available. Immunohistochemical studies were performed on the representative tissue sections from each case by the labelled streptovidin- biotin method. Sections for cyclin D1, p53, Bcl-2 immunostaining were pretreated in a microwave oven for 10 to 20 minutes in citrate buffer before immunostaining. The overnight incubation with NCL-cyclin D1-GM for cyclin D1, with clone DO-7 for p53, with clone 124 for bcl-2 was done. Mean follow-up was 24.1 months (range 2-84 months) after operation. Result: One hundred cases of lung cancers were composed of 56 cases of squamous cell carcinoma, 37 cases of adenocarcinoma, 5 cases of adenosquamous cell carcinoma, and 2 cases of large cell carcinoma. The 5-year survival was 32.1%. The positive expression rate of cyclin D1 was 35%, p53 was 56%, and bcl-2 was 17%. But there were no correlation between cyclin D1, p53, Bcl-2 protein expression and survival. Conclusion: These observation indicate that cyclin D1, p53, bcl-2 protein overexpression might be implicated in the oncogenesis of non-small cell lung carcinomas but they have no usefulness as a prognostic marker.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.29 no.4
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• pp.199-205
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• 2003

The p53 protein was discovered in 1979 as cellular 53-kD nuclear phosphoprotein bound to the large transforming antigen of SV40 virus. $P21^{WAF1/CIP1}$, which has been described as the critical downstream mediator of p53, is known to suppress DNA replication and arrest the G1 cell cycle by quaternary complex with cyclin D, cyclin-dependent kinase(CDK) and proliferating cell nuclear antigen(PCNA). In these days, some studies shows that the p21 can be induced by independent pathways. There are various reports about the expression of p21 (67%.82.4%) in oral squamous cell carcinoma. But these studies are mostly done in malignant tumor not in benign tumor. So we decided to study the expression of p21 in ameloblastoma and the relationship between p53 and p21 as a downstream mediator of p53 in ameloblastoma. We investigated the expression of p21 and p53 with the method of immunohistochemistry. We selected 30 cases of ameloblastoma tissue blocks (acanthomatous type: 5 cases, follicular type: 8 cases, plexiform type: 17 cases) imbedded in paraffin. We used 30 cases of normal gingival tissues and 30 cases of squamous cell carcinoma tissues (SCC) respectively and compared their results with those of ameloblastoma. We made slides with the streptavidin-biotin methods and used monoclonal antibody DO-7 (Novocastra, Newcastle, United Kingdom) as p53 antibody and monoclonal antibody M7202 (DAKO, California, U.S.A.) as p21 antibody. We used Pearson's correlation coefficient to analyse the relationship. The results were as follows: 1. p21 was expressed in ameloblastoma about 30% and this is lower than that of normal gingiva and SCC. 2. In normal gingiva and ameloblastoma, p21 expression was correlated with p53 expression. 3. In SCC, p21 were expressed about 83.3% and this is more than that of p53. But there was no correlation between p21 and p53 expression. We confirmed p21 expression and relation with p53 in ameloblastoma. But, to confirm the function of p21, more studies about p21 expression in malignant ameloblastoma and ameloblastic carcinoma are needed.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.20
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• pp.8651-8656
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• 2014

Purpose: To study the expression of angiogenin-2 (Ang-2) and its receptor Tie-2 in colorectal cancer and discuss the possible mechanisms behind this process. Materials and Methods: Using the streptavidin-peroxidase (SP) immunohistochemical method, paraffin sections from 100 colorectal cancer samples and 10 samples from tumor-adjacent normal tissue (> 2 cm from the edge of the gross tumor) were tested for protein expression of Ang-2, Tie-2, PI3K, and AKT. Reverse transcription-polymerase chain reaction and Western blots were further used to measure expression of the 4 genes and proteins in 20 freshly-resected colorectal cancer samples and tumor-adjacent normal tissues. Results: In colorectal cancer tissues, the expression of the Ang-2, Tie-2, PI3K, and AKT genes and their proteins was significantly higher than in tumor-adjacent normal tissues. Protein expression in poorly-differentiated adenocarcinoma was higher than that in well and moderately differentiated adenocarcinoma. According to Duke's classification, the protein expression in Stages C and D was significantly higher than that in Stages A and B. In the group with lymphatic metastasis, the protein expression was higher than that without lymphatic metastasis. Conclusions: In colorectal cancer, the expression of the Ang-2, Tie-2, PI3K, and AKT genes and their proteins is markedly higher than those in tumor-adjacent normal tissues. No correlation was observed between protein expression and gender, location, or histologic type. Correlations did exist between protein expression and differentiation level, stage of Duke's classification, and lymphatic metastasis; in colorectal cancer tissues with lower differentiation levels, higher stages of Duke's classification, and lymphatic metastasis, the expression of all 4 proteins was higher. The study of their expression patterns and relationships with aggression and metastasis will provide a valuable experimental foundation for assessing prognosis and targeted therapy of colorectal cancer.