• Bulletin of the Korean Chemical Society
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• v.31 no.11
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• pp.3259-3264
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• 2010

A label-free amperometric immunosensor has been proposed for the detection of myeloperoxidase (MPO) in human serum. To fabricate such an immunosensor, a composite film consisting of N,N-dimethylformamide (DMF), multiwall carbon nanotubes (MWCNTs) and 1-ethyl-3-methyl imidazolium tetrafluoroborate ($EMIMBF_4$) suspension was initially formed on a glassy carbon electrode (GCE). Then cerium dioxide ($CeO_2$) dispersed by chitosan was coated on the GCE. After that, MPO antibodies (anti-MPO) were attached onto the nano$CeO_2$ surface. With a noncompetitive immunoassay format, the antibody-antigen complex formed between the immobilized anti-MPO and MPO in sample solution. The immunosensor was characterized by cyclic voltammetry, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The factors influencing the performance of the immunosensor were studied in detail. Under optimal conditions, the current change before and after the immunoreaction was proportional to MPO concentration in the range of 5 to $300\;ng\;mL^{-1}$ with a detection limit of $0.2\;ng\;mL^{-1}$.

• Journal of fish pathology
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• v.12 no.1
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• pp.24-31
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• 1999

Optimization and standardization of solid phase enzyme immunoassay were done for the diagnosis of edwardsiellosis in fish. The analyzed degree of immobilized antibody on surface of solid phase with peroxidase saturation method showed the optimized result by using partially purified $50{\mu}g/ml$ of rabbit anti-E. tarda Edk-2 antibody in sodium bicarbonate buffer for overnight incubation to cover the surface of polystyrene beads. Optimized immunoreaction was observed in the treatment of $50{\mu}g/ml$ of biotin conjugated antibody followed extravidin-peroxidase diluted 1 : 2,000 in PBS. The detectable concentrations of the this method were $1{\times}10^5$ cells/ml and $1{\times}10^5$ cells/ml expressed as the source of antigen amount for EDTA extraction and heat extraction, respectively. High cross-reaction of solid phase ELISA with the prepared rabbit and-E. tarda Edk-2 was observed against E. tarda strains isolated from flounder suffering from edwardsiellosis in aquatic farms of Korea. It suggested that the potential of this solid phase of ELISA technique is very powerful for the application to different strains of E. tarda isolated in farms of many different areas.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.47-47
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• 2005

There are many different surface technologies currently applied for preparation of protein chips. However, it requires innovative surface chemistry for capture proteins to be immobilized on chip surface keeping their conformation and activity intact and their orientation right, while they bind tightly and densely in a given array spot. Proteogen has developed 'ProteoChip BP' coated with novel proprietary linker molecules $(ProLinker^{TM})$ for efficient and robust immobilizations of capture proteins by improving surface properties of molecular captures. It was demonstrated that $ProLinker^{TM}$ gave the best surface performance in preparation of protein microarray chip base plates among others currently available on the market. In particular, the $ProLinker^{TM}-based$ surface chemistry has demonstrated to provide excellent performance in preparation of 'Antibody Chip' for analysis of biomarkers as well as proteome expression profiles. The linker molecule has also shown to be well applicable for development of biosensors and micro-beads as well as protein microarray and nano-array. ProteoChip BP can be used either for preparation of high-density array by using a microarrayer or for preparation of 'Well-on-a-Chip' with low density array, which is better applicable for quantitative analysis of biomarkers or protein-protein interactions. The biomarker assay can be performed either by direct or sandwich methods of fluorescence immunoassay. Application of ProteoChip BP has been well demonstrated by the extensive studies of 1) tumor-marker assays, 2) new drug screening by using 'Integrin Chip' and 3) protein expression profile analysis. Some of experimental results will be presented.

• BMB Reports
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• v.36 no.3
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• pp.299-304
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• 2003

For deciphering the cyclic guanosine monophosphate (cGMP) signaling pathway, we employed chemical proteomics to identify the novel target molecules of cGMP. We used cGMP that was immobilized onto agarose beads with linkers directed at three different positions of cGMP. We performed a pull-down assay using the beads as baits on tissue lysates and identified 9 proteins by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry. Some of the identified proteins were previously known cGMP targets, including cGMP-dependent protein kinase and cGMP-stimulated phosphodiesterase. Surprisingly, some of the co-precipitated proteins were never formerly reported to associate with the cGMP signaling pathway. The competition binding assays showed that the interactions are not by nonspecific binding to either the linker or bead itself, but by specific binding to cGMP. Furthermore, we observed that the interactions are highly specific to cGMP against other nucleotides, such as cyclic adenosine monophosphate (cAMP) and 5'-GMP, which are structurally similar to cGMP. As one of the identified targets, MAPK1 was confirmed by immunoblotting with an anti-MAPK1 antibody. For further proof, we observed that the membrane-permeable cGMP (8-bromo cyclic GMP) stimulated mitogen-activated protein kinase 1 signaling in the treated cells. Our present study suggests that chemical proteomics can be a very useful and powerful technique for identifying the target proteins of small bioactive molecules.

• Journal of Biosystems Engineering
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• v.34 no.1
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• pp.50-56
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• 2009

Surface plasmon resonance (SPR) biosensor has been used to detect many biochemical reactions, because this label-free sensor has high sensitivity and rapid response. The reactions are monitored by refractive index changes of the SPR biosensor. Iprovalicarb is protective, curative, and eradicative systemic fungicide introduced by Bayer AG in 1999. It has potential far control of downy mildew infesting onion, cucumber, grape and melon, late blight infesting tomato and potato, and anthracnose infesting watermelon and pepper. It is strictly limited to the maximum residue limit. In this study, the applicability of a portable SPR biosensor (Spreeta, Texas instrument, TX, USA) to detect the iprovalicarb residue was examined. The sensor chip was adopted to detect the reaction of iprovalicarb to immobilized iprovalicarb-antibody. The binding of the iprovalicarb onto the biosensor surface was measured by change of the refractive index (RI). Characteristics of the sensor chip including specificity, sensitivity, stability, and reusability were analyzed. In calibration test for seven levels of iprovalicarb concentration (0.32 to 5,000 mg/L) with three replications, a Sigmoidal model with Hill function was obtained between relative RI value and the iprovalicarb concentration with R-square of 0.998. It took 30 minutes to complete a set of detecting assay with the SPR biosensor.

• Journal of Microbiology
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• v.42 no.2
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• pp.126-132
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• 2004

A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30$^{\circ}C$ until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30$^{\circ}C$ for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Topl0F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, com-pared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.

• Korean Journal of Clinical Pharmacy
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• v.8 no.1
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• pp.29-34
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• 1998

Murine CD38 is a 42 kDa type II glycoprotein expressed on cell surface of both B and T lymphocytes. CD38 is a multifunctional enzyme that catalyzes the formation and hydrolysis of cyclic adenosine diphosphoribose (cADPR): ADP-ribosyl cyclase activity of CD38 catalyzes the formation of cADPR from NAD and cADPR hydrolase activity of CD38 catalyzes the hydrolysis of cADPR to ADP-ribose (ADPR). And also, CD38 has the catalytic activity of NAD glycohydrolase (NADase) which catalyzes the hydrolysis of catalyzes the formation and hydrolysis of cyclic adenosine diphosphoribose (cADPR): ADP-ribosyl cyclase activity of CD38 catalyzes the formation of cADPR from NAD to ADPR. In this study, we attempted to purify CD38 from mouse lymphocytes by using the immobilized anti-CD38 monoclonal antibody. The single step immuno-affinity column chromatography resulted in homogeneous purification, showing a single protein of 42 kDa on a SDS polyacrylamide gel. We have investigated the effects of various inhibitors on the enzyme activities of the purified CD38. Cibacron blue (0.5 mM) inhibited all three enzyme activities of CD38, NADase, ADP-ribosyl cyclase and cADPR hydrolase activities. ADPR (2 mM) showed inhibitory effect on both cADPR hydrolase activity and NADase, but not on ADP-ribosyl cyclase activity. However, ATP (2 mM) inhibited only cADPR hydrolase activity. $Zn^{2+}$ (1 mM) showed similar inhibitory effect as that of ADPR, but activated cyclase activity These results suggest that CD38 has three different catalytic activity domains which might be differentially regulated by their specific inhibitors.

• Korean Journal of Animal Reproduction
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• v.16 no.2
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• pp.87-102
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• 1992

A rapid, sensitive, and specific enzyme-linked immuno-sorbent assay (ELISA) for bovine casein was developed. Biotinylated casein and peroxidase-conjugated avidin were used in the assay with antibody separated from yolks of immunized hens. Caseins were biotinylated with sulfo-N-hydroxy succinimido biotin and peroxi-dase-conjugated avidin bound the biotinylated casein which became bound to immobilized anti-body on a microplate. The antibodies were specific for bovine $\alpha$- and $\beta$-caseins, and their cross-reactivities with whey proteins, IgG, and serum albumin from bovine were not detectable by ELISA and Western blot. Various sensitivities ranging from 2ng/ml to 20${\mu}\textrm{g}$/ml of casein were achieved, and were controlled by adding vanous concentrations of the biotinylated casein. Parallelism was observed between standard and sample curves. The coefficients of variation of intra-assays and inter-assays from the most sensitive assay were 5.5 and 5.7%, respectively, at the 50% displacement. Casein contents of peripaturient milk samples showed that casein secretion rapidly increased 3d prepartum.

• Applied Chemistry for Engineering
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• v.22 no.5
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• pp.522-525
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• 2011

In this study, an established enzyme-linked immunosorbent assay and immuno-chromatography technique are combined to fabricate an immuno-strip sensor for the detection of S. aureus. The immuno-strip is manufactured by using four different functional membranes. The capture antibody is immobilized on the nitrocellulose membrane due to the high affinity and the capillary action through porous membranes induces a flow of sample. A colorimetric signal is appeared according to the enzyme reaction and is analyzed by the digital camera (qualitative analysis) and home-made image analysis software (quantitative analysis). Under the optimal conditions, samples with S. aureus in the range of $2.7{\times}10^4{\sim}2.7{\times}10^7CFU/mL$ can be detected by the colorimetric method within 30 min.

• KSBB Journal
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• v.22 no.6
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• pp.371-377
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• 2007

Polyhydroxyalkanoates (PHAs) are a family of microbial polyesters that can be produced by fermentation from renewable resources. PHAs can be used as completely biodegradable plastics or elastomers. In this paper, novel applications of PHAs in biosensor are described. A general platform technology was developed by using the substrate binding domain (SBD) of PHA depolymerase as a fusion partner to immobilize proteins of interest on PHA surface. It could be shown that the proteins fused to the SBD of PHA depolymerase could be specifically immobilized onto PHA film, PHA microbead, and microcontact printed PHA surface. We review the results obtained for monitoring the specific interaction between the SBO and PHA by using enhanced green fluorescent protein, red fluorescent protein, single chain antibody against hepatitis B virus preS2 surface protein and severe acute respiratory syndrome coronavirus surface antigen as model proteins. Thus, this system can be efficiently used for studying protein-protein and possibly protein-biomolecule interactions for various biotechnological applications.