研究業績集

学会発表2008年

タイトル

Development and Application of a New Assay System (CLEIA) for Hepatitis B Virus Core-Related Antigen (HBcrAg).

発表者

Hitoshi Kikuchi, Atsushi Kaneko and Sachiko Kitajima

学会名

AACC 2008

内容

Objects: Hepatitis B virus (HBV) is an important causative agent for liver disease such as chronic hepatitis, cirrhosis and hepatocellular carcinoma, and several markers are utilized for screening of HBV infection and for monitoring of clinical status of HBV-causative diseases. Recently, a new assay was performed for the detection of Hepatitis B core-related antigen (HBcrAg) consisting of HBV core antigen (HBcAg), HBeAg and 22kDa precore protein (p22cr) coded with precore/core gene. p22cr is a precore protein from aa -28 to at least aa 150, containing an uncleaved signal sequence and lacking a C-terminal ariginine-rich domain. We evaluated this assay system’s kit performance and clinical usefulness for monitoring of chronic hepatitis under treatment with nucleotide and/or nucleoside analogs such as lamivudine. Method: The assay system was the two-step sandwich CLEIA method using ferrite microparticles coated with 3 kinds of monoclonal antibodies against HBcrAg and 2 kinds of monoclonal antibodies coupled with alkaline phosphatase. Relative chemiluminescence intensity was measured, and HBcrAg concentration was calculated by a standard curve generated using recombinant pro-HBeAg (amino acids: 10 to 183 of precore/core gene product). HBcrAg concentration was expressed as units/mL (U/mL), which is defined as the immunoreactivity of 10 fg/mL of recombinant pro-HBeAg. CLEIA was performed on the fully automated Lumipulse f CLEIA analyzer (Fujirebio, Inc.) Serum samples underwent pretreatment with a solution containing 15% sodium dodecylsulfate at 60C for 30 min before assay, a process that is essential for HBcAg and HBeAg antibody degradation. For evaluation of clinical utility, 81 patients with chronic hepatitis, who received lamivudine therapy, were enrolled in this study, and serum samples were collected under informed consent. Serological markers for HBV, HBsAg, HBeAg, HBeAb and HBV DNA were also measured. Results: An appropriate calibration curve was obtained by HBcrAg standards, and the assay range was from 3.0 to 7.0 Log U/mL. Intra- and inter-assay coefficient of variation, (CV) was below 1.1 % and 1.1 %, respectively. The recovery of added HBcrAg standard to serum samples was more than 98.1 %. No interference of blood components was confirmed by adding free and conjugated forms of bilirubin, hemoglobin and lipids. In the clinical study, HBcrAg concentration was correlated with serum HBV DNA conc. in chronic hepatitis patients before lamivudine treatment. The levels of both HBcrAg and HBV DNA decreased after the start of lamivudine administration, but the level of HBcrAg decreased significantly more slowly than that of HBV DNA. In addition, the level of HBcrAg in chronic hepatitis B patients before the lamivudine treatment correlated with the incidence of lamivudine-resitant HBV, suggesting that the measurement of HBcrAg is useful as marker of occurrence of lamivudine-resistance. Conclusion: The HBcrAg CLEIA is a fully automated, reliable and sensitive system with a throughput of 120 tests per hour. HBcrAg under treatment with lamivudine is useful for monitoring of HBV activation and/or residual intrahepatic HBV.

タイトル

臨床検体を用いたG-tail assay法の確立

発表者

青木絵理子1)2)、嶋本 顕1)、丹羽敏博3)、鳩岡未沙子1)、頼岡徳在4)、田原栄俊1)
1)広島大院歯薬・細胞分子生物学研 2)日本科学技術振興機構 3)富士レビオ株式会社 4)広島大院歯薬・腎臓病制御学講座

学会名

日本薬学会第128年会

内容

【目的】染色体末端には、5’-TTAGGG-3’の繰り返し配列からなるテロメアDNAがある。そのほとんどは二本鎖であるが、その最末端にはG-tailと呼ばれる3’末端が数百塩基突出した一本鎖テロメアDNA部分が存在する。近年、このG-tailが染色体の安定性の維持においてきわめて重要であることがわかってきた。テロメア長の短縮と種々の老化関連疾患との関連を示す報告は多くあるが、G-tail長との関連の報告はない。これは、わずか数百塩基程度のG-tail長の測定が困難であることに起因するが、当研究室ではこの問題を解決したG-tail telomere HPA法という迅速、簡便かつ高感度なG-tail長の測定系の確立に世界で初めて成功した(Nat. Methods.,2005)。今回、この方法を臨床血液サンプルを用いた多検体処理するための改良を行い、老化関連疾患やがんなどのリスク評価を行う臨床診断に向けたG-tail telomere HPA法の確立を目的とした。
【方法】倫理的配慮のもと採取したヒト末梢血サンプルから、G-tail telomere HPA法に適した改良フェノールクロロホルム法により単核細胞DNAを抽出・精製した。精製したゲノムDNAを用いてG-tail telomere HPA法によるG-tail長並びにテロメア全長の測定を行った。また、比較のために従来よりテロメア長の測定法として利用されているSouthern blot法によるテロメア全長の測定も行った。
【結果および考察】G-tail telomere HPA法によるテロメア長の結果では、G-tail長はテロメア全長とほぼ相関していた。またG-tail telomere HPA法でテロメア全長の長かったサンプルではSouthern blot法でも長く、両方法により得られた相対的なテロメア全長及びG-tailの結果は非常によく相関していた。以上のように、臨床血液サンプルを用いたG-tail telomere HPA法の開発に成功した。

タイトル

Specificity and Affinity in Protein-Protein Interactions: From a Systems Biology to a Molecular Point of View

発表者

Pablo Carbonell

学会名

12th Annual International Conference on Research in Computational Molecular Biology RECOMB 2008

内容

We compiled a dataset of protein-protein interactions with structural information from the yeast interactome. For each interaction we were able to find a representative structure of the complex. Using the structural information, we identified hub proteins, clustered their binding sites, and classified them into singlish-interface hubs (involving only one interface for binding) and multi-interface hubs (involving more than one interface for binding). Furthermore, we counted the number of nonredundant partners interacting through each interface. Specific binding sites were defined as those binding sites interacting with only one partner, whereas promiscuous binding sites interact with more than one partner.
We calculated hydrophobic patches on the protein hub surfaces [1], seeing a clear tendency for the multi-interface hubs to have on average a larger number of patches distributed over their surfaces. This result suggests that the patches analysis could be used to identify multi-interface hubs, which, according to previous studies, are more likely essential for cellular viability [2].
The affinity of the interactions was estimated by calculating the binding free energy of each interaction. Our estimations were based on the rigid structure of the complex, and therefore large entropic contributions such as ordering of disordered binding sites upon binding were not considered. Consequently, in our estimations those interactions associated with disordered binding sites were found to require a high enthalpic contribution to compensate the entropic cost of binding. Based on this finding, and in order to estimate the binding free energy more precisely, we restricted our affinity analysis to protein-protein interactions involving ordered binding sites.We identified residues essential for binding (hotspots) for each protein interface [3], and analyzed how they were distributed across the set of different interactions. Hotspots that were found in more than one interaction might play an essential role for determining affinity, whereas those associated with single interactions could be responsible for binding site specificity.

タイトル

Novel Detection Scheme for Optical Biosensing Using Whispering Gallery Modes in Clusters of Dielectric Particles

発表者

Alexandre FRANCOIS, Michael HIMMELHAUS

学会名

SPIE Photonics West 2008

内容

We present a novel concept for an optical biosensor based on Whispering Gallery Mode (WGM) excitations in clusters of spherical microresonators. Compared to single particles, clusters of microresonators offer the advantage to be easier detected due to their higher radiative emission power. Our results demonstrate that clusters of microresonators can be used for the detection of biomolecules in the same way as single spheres. With the actual choice of materials, we achieved a mass sensitivity limit of 100 fg, which is 30 times more sensitive than that of state-of-the-art WGM biosensors. Further improvements of the performance can be achieved by increasing the refractive index of the microresonators, by reducing their diameter and/or by operation in lasing mode. These modifications either reduce the linewidths of the WGM or increase their wavelength shift associated with the adsorption of biomolecules, and thus improve sensitivity as well as detection limit of the system.

タイトル

Modular architecture of protein structures and allosteric communications: potential implications to signaling proteins and regulatory linkages

発表者

Alexandre FRANCOIS, Michael HIMMELHAUS

学会名

SPIE Photonics West 2008

内容

Whispering gallery modes (WGM) in fluorescent dielectric microcavities have recently become an attractive alternative to state-of-the-art label-free optical biosensors due to their high sensitivity to molecular adsorption and their ease of operation under a variety of environmental conditions. In particular the true microscopic dimension of the sensor as well as its purely radiative control without any need for external coupling opens new opportunities for label-free biosensing on microscopic scale.
While these are obvious advantages, a direct comparison of the performance of WGM biosensors with well-established techniques of known high sensitivity, such as surface plasmon resonance sensors, has not been undertaken to date, thus obscuring the opportunities of the newly rising approach.We have therefore studied the performance of both WGM biosensors and a commercial SPR sensor using a selection of specifically and non-specifically binding biomolecules in-situ and under same conditions. The performance of the two techniques is compared in view of the efficiency and sensitivity towards detection of both model interaction pairs (e.g. Avidin/biotin) and specific interaction pairs such as antigen-antibody with varying degrees of interaction affinities.

タイトル

Enhancing Surface plasmon detection of biomolecular interactions through use of nanostructured interfaces

発表者

Michael HIMMELHAUS

学会名

SPIE Photonics West 2008

内容

In this work, we aim at enhancing the sensitivity of surface plasmon resonance sensors towards the detection of biomolecule interactions by means of nanopatterning of the sensor surface. Use of nanostructured interfaces in combination with SPR is a promising step towards realizing biosensors with high efficiency and sensitivity. Nanopatterned surfaces enable multi-dimensional control over the behavior of surface-immobilized receptor molecules. In this talk we will first show that by means of confinement inducing nanopatterns consisting of fouling and non-fouling areas an enhancement in activity of surface immobilized antibodies can be achieved in addition to a substantial increase in their areal density. We further explore surfaces such as nanoparticle arrays that not only provide means of higher surface per unit area, but also allow for means of alleviating steric stress associated with the binding events. Patterns with contrast in topography and surface chemistry are compared for their influence on behavior of immobilization of antibodies and the activity of immobilized antibodies using surface plasmon resonance based detection of binding events. The results are compared against standard ELISA assays performed on the nanostructured surfaces.

タイトル

Optical Cavity Mode Excitations in Metal-Coated Microspheres

発表者

Michael HIMMELHAUS

学会名

SPIE Photonics West 2008

内容

We present some recent results of our work on cavity mode excitations in metal-coated microspheres. In contrast to the well-known whispering gallery modes (WGM) of dielectric particles, metal-coated dielectric microspheres also allow for excitation of modes in radial direction, the so-called Fabry-Perot modes (FPM) [1]. One hurdle of such excitation is the proper adjustment of the reflectivity of the metallic coating, which either causes a low quality factor of the modes in case of insufficient thickness, however, shields the inner cavity from outside excitation otherwise. The talk will present a novel concept on how such intricacies may be overcome by proper selection of excitation wavelength and materials choice, and will demonstrate that FPM modes may be excited in microcavities with diameters down to 1 mm. Potential applications of such small metallic microcavities are related to the development of optical point sources, microscopic lasers, and to nonlinear nano-photonics.