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Paek, Sung-Ho,Cho, Il-Hoon,Seo, Sung-Min,Kim, Dong-Hyung,Paek, Se-Hwan Royal Society of Chemistry 2011 The Analyst Vol.136 No.20
<P>To effectively control diabetes, a method to reliably measure glucose fluctuations in the body over given time periods needs to be developed. Current glucose monitoring systems depend on the substrate decomposition by an enzyme to detect the product; however, the enzyme activity significantly decays over time, which complicates analysis. In this study, we investigated an alternative method of glucose analysis based on antigen–antibody binding, which may be active over an extended period of time. To produce monoclonal antibodies, mice were immunized with molecular weight (<I>M</I><SUB>W</SUB>) 10K dextran chemically conjugated with keyhole limpet hemocyanin. Since dextran contains glucose molecules polymerized <I>via</I> a 1,6-linkage, the produced antibodies had a binding selectivity that could discriminate biological glucose compounds with a 1,4-linkage. Three antibody clones with different affinities were screened using the <I>M</I><SUB>W</SUB> 1K dextran–bovine serum albumin conjugates as the capture ligand. Among the antibodies tested, the antibody clone Glu 26 had the lowest affinity (<I>K</I><SUB>A</SUB> = 3.56 × 10<SUP>6</SUP> M<SUP>−1</SUP>) and the most rapid dissociation (<I>k</I><SUB>d</SUB> = 1.17 × 10<SUP>−2</SUP> s<SUP>−1</SUP>) with the polysaccharide immobilized on the solid surfaces. When glucose was added to the medium, the sensor signal was inversely proportional to the glucose concentration in a range between 10 and 1000 mg dL<SUP>−1</SUP>, which covered the clinical range. Under the optimal conditions, the response time was about 3 min for association and 8 min for dissociation based on a 95% recovery of the final equilibrium.</P> <P>Graphic Abstract</P><P>An antibody produced for CGM was specific to the 1,6-glycosidic linkage of polysaccharides and the low multiple binding, discriminating from Con A. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c1an15338b'> </P>
Site-directed Immobilization of Antibody onto Solid Surfaces for the Construction of Immunochip
Paek, Se-Hwan,Cho, Il-Hoon,Paek, Eui-Hwan,Lee, Haewon,Park, Jeong-Woo The Korean Society for Biotechnology and Bioengine 2004 Biotechnology and Bioprocess Engineering Vol.9 No.2
The performance of an immuno-analytical system can be assessed in terms of its analytical sensitivity, i.e., the detection limit of an analyte, which is determined by the amount of analyte molecules bound to the capture antibody that has been immobilized onto a solid surface. To increase the number of the binding complexes, we have investigated a site-directed immobilization of an antibody that has the ability to resolve a current problem associated with a random arrangement of the insolubilized immunoglobulin. The binding molecules were chemically reduced to produce thiol groups that were limited at the hinge region, and then, the reduced products were coupled to biotin. This biotinylated antibody was bound to a streptavidin-coated surface via the streptavidin-biotin reaction. This method can control the orientation of the antibody molecules present on a solid surface and also can significantly reduce the possibility of steric hindrance in the antigen-antibody reactions. In a two-site immunoassay, the introduction of the site-directly immobilized antibody as the capture enhanced the sensitivity of analyte detection approximately 10 times compared to that of the antibody randomly coupled to biotin. Such a novel approach would offer a protocol of antibody immobilization in order for the possibility of constructing a high performance immunochip.
최훈 ( Hoon Choi ),김형수 ( Hyung Soo Kim ),서정혁 ( Jung Hyuck Suh ),백옥진 ( Ock Jin Paek ),강영운 ( Young Woon Kang ),이준구 ( Joon Goo Lee ),봉영훈 ( Young Hoon Bong ),곽지연 ( Ji Yeon Kwak ),이승일 ( Seung Il Lee ),신민홍 ( 한국환경농학회 2013 한국환경농학회 학술대회집 Vol.2013 No.-
This study was carried out to survey the levels of mercury in root vegetables and assess dietary exposure / risk to the Korean population health. Various samples including Panax ginseng C.A mayer, Codonopsis lanceolata, and Platycodon granditloum were collected from markets across Korea. The concentrations of mercury were measured using automatic mercury analyzer. The analytical and sampling methods in this study were full validated. The mercury content in root vegetables was determined below 100 μg/kg (maximum residue of mercury for salt established in Korea). For risk assessment, probable daily intake was calculated and compared with PTWI (tolerable weekly intake) established by JECFA. The level of overall exposure to mercury for Korean through root vegetables was below 1% of the recommended JECFA levels, indicating of little possibility of risk. Whereas, MFDS was conducted to assess the dietary exposure to mercury from food intake, based on several reports regarding mercury published by MFDS in the 2000s. The mean and 95th percentile for exposure to dietary mercury were 4.29 and 12.48 μg/day, corresponding to 13.6% and 39.7% of PTWI, respectively.
Toll-like receptor-based immuno-analysis of pathogenic microorganisms.
Cho, Il-Hoon,Jeon, Jin-Woo,Paek, Sung-Ho,Kim, Dong-Hyung,Shin, Hee-Sung,Ha, Un-Hwan,Seo, Sung-Kyu,Paek, Se-Hwan American Chemical Society 2012 ANALYTICAL CHEMISTRY - Vol.84 No.22
<P>In this study, a novel mammalian cell receptor-based immuno-analytical method was developed for the detection of food-poisoning microorganisms by employing toll-like receptors (TLRs) as sensing elements. Upon infection with bacterium, the host cells respond by expressing TLRs, particularly TLR1, TLR2, and TLR4, on the outer membrane surfaces. To demonstrate the potential of using this method for detection of foodborne bacteria, we initially selected two model sensing systems, expression of TLR1 on a cell line, A549, for Escherichia coli and TLR2 on a cell line, RAW264.7, for Shigella sonnei (S. sonnei). Each TLR was detected using antibodies specific to the respective marker. We also found that the addition of immunoassay for the pathogen captured by the TLRs on the mammalian cells significantly enhanced the detection capability. A dual-analytical system for S. sonnei was constructed and successfully detected an extremely low number (about 3.2 CFU per well) of the pathogenic bacterium 5.1 h after infection. This detection time was 2.5 h earlier than the time required for detection using the conventional immunoassay. To endow the specificity of detection, the target bacterium was immuno-magnetically concentrated by a factor of 50 prior to infection. This further shortened the response to approximately 3.4 h, which was less than half of the time needed when the conventional method was used. Such enhanced performance could basically result from synergistic effects of bacterial dose increase and subsequent autocrine signaling on TLRs' up-regulation upon infection with live bacterium. This TLR-based immuno-sensing approach may also be expanded to monitor infection of the body, provided scanning of the signal is feasible.</P>
백옥진 ( Ock Jin Paek ),김형수 ( Hyung Soo Kim ),서정혁 ( Jung Hyuck Suh ),강영운 ( Young Woon Kang ),이준구 ( Joon Goo Lee ),봉영훈 ( Young Hoon Bong ),최훈 ( Hoon Choi ),곽지연 ( Ji Yeon Kwak ),이승일 ( Seung Il Lee ),박기훈 ( 한국환경농학회 2013 한국환경농학회 학술대회집 Vol.2013 No.-
Alternaria toxins haver been found to be natural food contaminants in grains, sunflowers seeds, and some visibly decayed fruits in many countries. Their natural occurrence in cereals has been reported in different countries. Several studies have reported the relevance of the this genus in crop. However, there are no studies on the method validation of alternaria toxin in cereals in our contry. A simple and sensitive analytical method based on HPLC with PDA and mobile phases using 0.1% phosphoric acid and acetonitrile was developed for simultaneous determination of alternaria toxins. A method validation for the determination of alternaria toxins in cereals was validated. In short, the method is as follows : A test portion of a sample is extracted with a mixture of methanol/water(90/10, v/v). This raw extract is then diluted, filtered, and applied to an SPE column. After washing and elution with acetonitrile, the elute is evaporated to dryness. After toxins in the dry residue in mobile phase are injected into a high performance liquid chromatography, and detected and quantified by PDA. For alternaria toxins in the cereals, Recovery test, calibration curves (Linearity), LOD and LOQ were successfully confirmed and Reproducibility relative standard deviations(RSDR) and Repeatability relative standard deviations(RSDr) for cereals samples were below 15% for the spiked cereals at 100 ug/kg. Since all these parameters lie well within the acceptable range set forth in EU mycotoxin method validation legislation. This method is The proposal method is sensitive, repeatable and rapid enough to apply to officially routine inspection of agricultural products including cereals.