• Applied Chemistry for Engineering
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• v.35 no.4
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• pp.335-340
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• 2024

In this paper, we synthesized organic and inorganic hybrid materials to introduce antibody functionality to UIO-66 and incorporated them into a surface plasmon resonance (SPR) assay to enhance the sensitivity of detecting small molecules such as oxytocin. A biological marker peptide called oxytocin may help in the diagnosis of heart failure, Alzheimer's disease, and cancer. To detect oxytocin at concentrations as low as a few femtomole (fM), we developed a surface sandwich assay utilizing a pair of oxytocin-specific antibodies for enhancing selectivity and one of metal organic frameworks [e.g., UiO-66-(COOH)₂] possessing high porosity and surface-area as a signal amplifier. Initially, real-time SPR assays were used to confirm that each selected oxytocin-specific antibody binds strongly to oxytocin and to different binding sites on oxytocin. One of these antibodies (e.g., anti-OXT[OTI5G4]) was immobilized on the surface of a thin gold chip. Upon sequential injecting of oxytocin and the other antibody (e.g., anti-OXT[4G11]) conjugated to UiO-66-(COOH)₂ onto the surface to form the surface sandwich complex of anti-OXT[OTI5G4]/oxytocin/UiO-66-(COOH)₂-anti-OXT[4G11]), SPR changes, which varied with oxytocin concentration, were then measured in real time. The results demonstrated that sensitivity was amplified by over a million-fold compared to assays without UiO-66-(COOH)₂, enabling oxytocin detection down to approximately 10 fM.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.2
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• pp.143-146
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• 2004

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the hexahistidine-ubiquitin-tagged human parathyroid hormone fragment (His$\sub$6/-Ub-hPTHF(1-34)) expressed in Escherichia coli. The hexahistidine-specific antibody was immobilized on a thin gold film coated with ProLinker$\^$TM/ B, a novel calixcrown derivative with a bifunctional coupling property that permits efficient immobilizaton of capture proteins on solid matrices. The soluble and insoluble fractions of an E. coli cell lysate were spotted onto the antibody-coated gold chip, which was then washed with buffer (pH 7.4) solution and dried. SPR imaging measurements were carried out to detect the expressed His$\sub$6/-Ub-hPTHF(1-34). There was no discernible protein image in the uninduced cell lysate, indicating that non-specific binding of contaminant proteins did not occur on the gold chip surface. It is expected that the approach used here to detect affinity-tagged recombinant proteins using an SPR imaging technique could be used as a powerful tool for the analyses of a number of proteins in a high-throughput mode.

• IMMUNE NETWORK
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• v.3 no.1
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• pp.8-15
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• 2003

Background: CD30 is a member of TNF receptor family and expressed on lymphocytes and other hematopoietic cells following activation as well as Hodgkin and Reed-Sternberg cells in Hodgkin's lymphoma. In this study, CD30-mediated regulation of cell adhesion molecule expression on normal activated mouse T cells was investigated. Methods: Mouse T cells were activated with anti-CD3 antibody for induction of CD30, which was cross-linked by immobilized anti-CD30 antibody. Results: High level of CD30 expression on T cells was observed on day 5, but only little on day 3 even under culture condition resulting in an identical T cell proliferation, indicating that CD30 expression requires a prolonged stimulation up to 5 days. Cross-linking of CD30 alone altered neither proliferation nor apoptosis of normal activated T cells. Instead, CD30 appeared to promote cell adherence to culture substrate, and considerably upregulated ICAM-1 and, to a lesser extent, ICAM-2 expression on activated T cells, whereas CD2 and CD18 (LFA-1) expression was not affected. None of cytokines known as main regulators of ICAM-1 expression on tissue cells (IL 4, $IFN{\gamma}$ and $IFN{\alpha}$) enhanced ICAM-1 expression in the absence of CD30 signals. On the other hand, addition of $NF-{\kappa}B$ inhibitor, PDTC (0.1 mM) completely abrogated the CD30-mediated upregulation of ICAM-1 expression, but not CD2 and ICAM-2 expression. Conclusion: This results support that CD30 upregulates ICAM-1 expression of T cell and such regulation is not mediated by higher cytokine production but $NF-{\kappa}B$ activation. Therefore, CD30 may play important roles in T-T or T-B cell interaction through regulation of ICAM-1, and -2 expression.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2493-2496
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• 2010

Erythropoietin (EPO) is mainly produced in kidney and stimulates erythropoiesis. The use of recombinant EPOs for doping is prohibited because of its performance enhancing effect. This study investigated whether biosimilar EPOs could be differentiated from endogenous one by iso-electro-focusing plus double blotting and SDS-PAGE for antidoping analysis. The established method was validated with positive control urine. The band patterns were reproducible and meet the criteria, which was made by world anti doping agency (WADA). Isoelectric focusing was conducted in pH range 2 to 6. Recormon (La Roche), Aropotin (Kunwha), Epokine (CJ Pharm Co.), Eporon (Dong-A), Espogen (LG Life Sciences), and Dynepo (Shire Pharmaceuticals) were detected in basic region. All biosimilars showed discriminative isoelectric profiles from endogenous EPO profiles, but they showed different band patterns with the reference one except Epokine (CJ Pharm Co.). Next, SDS-PAGE of biosimilar EPOs resulted in different molecular weight patterns which were distributed higher than endogenous EPO. Commercial immune assay kit as an immune affinity purification tool and immobilized antibody coated magnetic bead were tested for the purification and concentration of EPO from urinary matrix. The antibody-coated magnetic bead gave better purification yield. The IEF plus double blotting and SDS-PAGE with immunoaffinity purification method established can be used to discriminate biosimilar EPOs from endogenous EPO.

• Journal of Biosystems Engineering
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• v.32 no.6
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• pp.440-447
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• 2007

In this study, a biosensor was developed using an enzyme-linked immunosorbent assay (ELISA) to rapidly measure the fungicide iprovalicarb residues in agricultural products. The biosensor was designed to include micro-pumps and solenoid valves for fluid transport, a spectrophotometer cuvet as a reaction chamber, a photodiode with a light-emitting diode for optical density measurement, and a control microcomputer to implement assay. The rate of change in optical density of the cuvet was read as final signal output. Micro-pumps were evaluated to investigate their delivery capability, the highest values of the error and the coefficient of variation were 4.3% and 4.6% respectively. As the incubation period was reduced from 15 minutes to 11 minutes to shorten the total processing time, the sensor sensitivity was decreased as the antibody dilution ratio was reduced to a half. The maximum usable period of the coated cuvet was found to be two days with 1% error limit. To predict the concentration of the iprovalicarb residue in agricultural products, a linear calibration model was obtained with r-square values of 0.992 for potato and 0.985 for onion. In validation test for the samples of potatoes and onions against the high performance liquid chromatography, very high correlation values were obtained as 0.996 and 0.993 respectively. Using the cuvet immobilized with antigen, it took 21-minutes for the biosensor to complete the measuring process of the iprovalicarb residues.

• Bulletin of the Korean Chemical Society
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• v.31 no.5
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• pp.1215-1218
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• 2010

We demonstrate the possible application of the sandwich type surface-enhanced Raman scattering (SERS) immunoassay using antigen-antibody binding for detection of prostate-specific antigen (PSA) in cancer cells. In this sandwich type of SERS immunoassay, to capture antigens onto the immobilized layer of antibodies on the gold substrate we prepared the monolayer of gold nanoparticles on the APTMS-derivatized surface of a glass slide by using the SAM technique. This sandwich type of SERS immunoassay in which antigens on the substrate specifically capture antibodies on a Raman reporter (DSNB coated gold nanoparticles with R6G) could successfully detect PSA at low levels. A strong SERS spectrum of Raman reporter was observed only with a substrate in which PSA is present.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.468-472
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• 2003

Recently, genetic engineering techniques have been used to display various heterologous peptides and proteins (enzyme, antibody, antigen, receptor and fluorescence protein, etc.) on the yeast cell surface. Living cells displaying various enzymes on their surface could be used repeatedly as 'whole cell biocatalysts' like immobilized enzymes. We constructed a yeast based whole cell biocatalyst displaying T. reesei cellobiohydrolase I (CBH I ) on the cell surface and endowed the yeast-cells with the ability to degrade cellulose. By using a cell surface engineering system based on ${\alpha}-agglutinin,$ CBH I was displayed on the cell surface as a fusion protein containing the N-terminal leader peptide encoding a Gly-Ser linker and the $Xpress^{TM}$ epitope. Localization of the fusion protein on the cell surface was confirmed by confocal microscopy. In this study, we report on the genetic immobilization of T. reesei CBH I on the S. cerevisiae and hydrolytic activity of cell surface displayed CBH I.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.19-19
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• 2005

This paper reports a novel magnetic force-based microfluidic immunoassay using microbeads and magnetic nanoparticles. The magnetic force-based immunoassay was devised first and successfully applied to detect the rabbit IgG as the model analyte of microfluidic sandwich immunoassay. The microchannels were fabricated by poly(dimethysiloxane) (PDMS) molding processes and bonded on a slide glass by plasma treatment. At the part of the inlet, sample solution was hydrodynamically focused. The focused microbeads of sample solution were flowed through the 150 ${\mu}m$ width channel of outlet. However, when the microbeads are conjugated with the superparamagnetic nanoparticles under the applied magnetic fields, they will switch their flow path and flow through the 95 ${\mu}m$ width channel of outlet. The movements of microbeads conjugated with magnetic nanoparticles were demonstrated by magnetic field $gradients.^{1)}$ High magnetic field gradients using micro electromagnets could be applied to this detection method for high sensitivity and lower detection limit. In addition, the multiplexed $immunoassay^{2)}$ using an encoded microbead which is immobilized with a certain antibody could be possible using this detection principle.

• Journal of Powder Materials
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• v.16 no.3
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• pp.180-184
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• 2009

In this paper, the electrochemical non-enzyme immunosensor has been developed for the determination of salmonella antigen, using inverse voltammetry. For the estimation of salmonella antigen concentration, the $Fe_3O_4$ nanoparticles synthesized by microemulsion method were conjugated with salmonella antigen. Then, the immunocomplex between antibody immobilized on the transducer surface and antigen containing a magnetic nanoparticles was formed. From the linear relationship between the reduction peak current of Fe(III) and salmonella antigen concentration, it is suggested that the electrochemical non-enzyme biosensor is applicable to detect salmonella antigen in the concentration range of $10^1-10^5$ CFU/ml.

• IMMUNE NETWORK
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• v.4 no.2
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• pp.116-122
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• 2004

Background: LIGHT (TNFSF14) is a member of tumor necrosis factor superfamily and is the ligand for TR2 (TNFRSF14/HVEM). LIGHT is known to have proinflammatory roles in atherosclerosis. Methods: To find out the expression pattern of LIGHT in atherosclerotic plaques, immunohistochemical analysis was performed on human carotid atherosclerotic plaque specimens. LIGHT induced atherogenic events using human monocytic cell line THP-1 were also investigated. Results: Immunohistochemical analysis revealed expression of LIGHT and TR2 in foam cell rich regions in the atherosclerotic plaques. Double immunohistochemical analysis further confirmed the expression of LIGHT in foam cells. Stimulation of THP-1 cells, which express TR2, with either recombinant LIGHT or immobilized anti-TR2 monoclonal antibody induced interleukin-8 and matrix metalloproteinase(MMP)-9. Electrophoretic mobility shift assay demonstrated that LIGHT induces nuclear localization of transcription factor, nuclear factor $(NF)-{\kappa}B$. LIGHT induced activation of MMP-9 is mediated by $NF-{\kappa}B$, since treatment of THP-1 cells with the $NF-{\kappa}B$ inhibitor PDTC (pyrrolidine dithiocarbamate) completely blocked the activation of MMP-9. Conclusion: These data indicate that LIGHT is expressed in foam cells in atherosclerotic plaques and is involved in atherogenesis through activation of pro-atherogenic cytokine IL-8 and destabilization of plaque by inducing matrix degrading enzyme.