投稿論文2010年｜研究業績集｜活動紹介

投稿論文2010年

タイトル

Novel monoclonal antibodies recognizing the active conformation of epidermal growth factor receptor

著者

Nobuyuki Ise , Kazuya Omi , Kyoko Miwa , Hideo Honda , Shigeki Higashiyama 1) and Katsutoshi Goishi
1) Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Ehime 791-0295, Japan

雑誌名

Biochemical and Biophysical Research Communications 394 (3), 2010, p685-690

内容

The precise regulation of epidermal growth factor receptor (EGFR) is crucial for its function in cellular growth control. Although many antibodies against EGFR have been developed and used to analyze its regulation and function, it is not yet easy to analyze activated EGFR specifically. Activated EGFR has been mainly detected by its phosphorylation state using anti-phospho-EGFR and anti-phosphotyrosine antibodies. In the present study, we have established novel monoclonal antibodies which recognize the activated EGFR independently of its phosphorylation. Our antibodies detected active state of EGFR in immunoprecipitation and immunofluorescence, by recognizing the epitopes which are exposed through the conformational change induced by ligand-binding. Furthermore, we found that our antibodies preferentially detected the conformation of constitutively active EGFR mutants found in lung cancer cell lines. These results indicate that our antibodies may become novel research and diagnostic tools for detecting and analyzing the conformation of active EGFR in various cells and tissues.

タイトル

Optical Sensors Based on Whispering Gallery Modes in Fluorescent Microbeads: Response to Specific Interactions

著者

Michael Himmelhaus , Sivashankar Krishnamoorthy , and Alexandre Francois

雑誌名

Sensors 2010,10,1-x manuscripts; doi:10.3390/s90x0000x

内容

Whispering gallery modes (WGMs) in surface-fixated fluorescent polystyrene microbeads are studied in view of their capability of sensing the formation of biochemical adsorption layers on their outer surface with the well-established biotin-Streptavidin specific binding as the model system. Three different methods for analysis of the observed shifts in the WGM wavelength positions are applied and used to quantify the adsorbed mass densities, which are then compared with the results of a comparative surface plasmon resonance (SPR) study.

タイトル

テロメアGテール長による個人疾患リスク診断システムの開発

著者

田原栄俊 1), 嶋本顕 1), 丹羽敏博, 青木絵里子 2)
1）広島大学, 2）JST（独立行政法人　科学技術振興機構）

雑誌名

育成研究：JSTイノベーションプラザ広島平成19年度採択課題

内容

テロメアG-tail HPAの技術を用い、基礎研究サンプルや臨床サンプルのGテール長を、簡便かつ高感度に測定できるシステムを開発した。キットの作成を行い、装置による自動測定法を確立した。本技術は、これまでのテロメア長測定に必須であったDNAの電気泳動、遠心分離、アイソトープなどを全く必要とせず、同一チューブ内で反応・検出することができる画期的な測定法である。

タイトル

Development of a sphingosylphosphorylcholine detection system using RNA aptamers.

著者

Katsunori Horii (a), Kazuya Omi , Yoshihito Yoshida (a), Yuka Imai , Sakai Nobuya (a), Asako Oka, Hiromi Masuda (a), Makio Furuichi (a), Tetsuji Tanimoto and Iwao Waga (a)
(a)VALWAY Technology Center, NEC Soft, Ltd.,

雑誌名

Molecules.15(8), 2010, p5742-55

内容

Sphingosylphosphorylcholine (SPC) is a lysosphingolipid that exerts multiple functions, including acting as a spasmogen, as a mitogenic factor for various types of cells, and sometimes as an inflammatory mediator. In this report, we have generated RNA aptamers that bind to SPC with high affinity using an in vitro selection procedure and developed an enzyme-linked aptamer assay system using the minimized SPC aptamer that can successfully distinguish SPC from the structurally related sphingosine 1-phosphate.

タイトル

Analytical evaluation of a high-molecular-weight (HMW) adiponectin chemiluminescent enzyme immunoassay.

著者

Suguru Hayama，Tomoko Higuchi，Hideo Miyakoshi，Yasuko Nakano 1)
1）昭和大学薬学部

雑誌名

Clinica Chimica Acta 411, 2010, p2073-2078

内容

We prepared new assay reagents based on chemiluminescent enzyme immunoassay (CLEIA) on a fully-automated analyzer system using the same IH7 monoclonal antibody as for ELISA as solid-phase and detection antibodies (CLEIA/cartridge-type and CLEIA/bottle-type), because the conventional ELISA method requires complicated and lengthy assay procedures to obtain assay results. As results, the assay range of both CLEIA reagents were from 0.20 to 15.00 μg/mL, and lower limit of detection and quantification were lower than 0.0928 and 0.1346 μg/mL in CLEIA/cartridge-type and in CLEIA/bottle-type reagents, respectively. A good correlation was observed between both reagents, the imprecision test as % of coefficient variation in both reagents were less than 3.3%, and recovery test showed the range from 100% to 109%. HMW adiponectin concentration measured by CLEIA reagents was approximately half that measured by the previous ELISA because of reevaluation using freshly and highly purified HMW adiponectin standard. According to these results, the newly prepared CLEIA reagents are robust and adequate and can be used for the measurement of HMW adiponectin in the clinical laboratory.

タイトル

インタクトI型プロコラーゲン-N-プロペプチド（P1NP）

著者

鉄本融 1)，和田直子
1) 株式会社テイエフビー

雑誌名

Medical Technology，39(1)，2011，p30-32

内容

インタクトI型プロコラーゲン-N-プロペプチド（P1NP）は、骨芽細胞で合成、分泌されたI型プロコラーゲンがN―ペプチダーゼの作用により切断・放出される代謝産物で骨形成マーカーに分類される。骨形成の過程でもっとも早期に産生されること、薬剤投与などで血中濃度の変化が認められることから、骨粗鬆症などの診療、薬剤効果判定に有用である。本項では、試薬の測定原理、検体の安定性、基準値および治療による測定値の変動を紹介。

タイトル

I型コラーゲン架橋C-テロペプチド(CTX)

著者

和田直子

雑誌名

Medical Technology，39(1)，2011，p40-43

内容

βクロスラプス(CTX)は腎や肝での代謝を受けずに血中、尿中に排泄されるこれ以上分解されることのない最小単位のペプチドで骨吸収マーカーに分類される。現在、血液又は尿を用いる2種類のキットが市販されている。骨吸収抑制薬の効果判定又は経過観察に保険適用されており、他項目とのしばりはない。本項では、精度の高い結果を得るための各キットの検査精度や検体採取条件、また、国内外の臨床知見を最新の話題に基づいて紹介。

タイトル

Development of a particle agglutination method with soluble virus receptor for identification of poliovirus

著者

Minetaro Arita 1), Souji Masujima, Takaji Wakita 1), and Hiroyuki Shimizu 1)
1) Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan

雑誌名

Journal of Clinical Microbiology Aug.2010, p.2698-2702

内容

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples of acute flaccid paralysis (AFP) cases. In this study, we have developed a particle agglutination (PA) method with a soluble human PV receptor (hPVR) in a form of immunoadhesin (PVR-IgG2a) for the simple and rapid identification of PV. Sensitized gelatin particles with PVR-IgG2a showed specific agglutination with the culture fluid of PV-infected cells within 2 h of reaction by a one-step procedure. Detection limits for type 1, 2, and 3 PV(Sabin) strains were 1.5 x 10⁶ CCID₅₀, 5.3 x 10⁵ CCID₅₀, and 9.1 x 10⁵ CCID₅₀, respectively. Wild type PVs and PV isolates from acute flaccid paralysis cases examined were correctly identified with this PA method except some samples with a mixture of different serotypes of PVs, where minor population of PV was failed to be detected. These results suggest that this PA method is useful for the simple and rapid identification of PV although the sensitivity was not high enough to detect minor population of PV (less than 1/10 of the major population) in mixed PVs.

タイトル

ルミパルスメソテリンの基礎性能評価

著者

中町衛，桑原明子，村上弘，皆川英孝，森山和重

雑誌名

医学と薬学65(2)，2011，p269-277

内容

ルミパルスメソテリンの基礎評価を実施した。感度、正確性、再現性は良好な成績であった。希釈直線性は10倍まで問題なかった。対照品のMESOMARKと相関は、傾き1.07、相関係数0.999と良好であった。生体物質の影響は認められなかった。参考正常値は1.5nmoｌ/Lと算出された。悪性中皮腫の診断マーカーが他に存在しないことや、画像診断のみでは診断が難しい症例が存在するため、ルミパルスメソテリンは中皮腫の診断において有用と考えられた。

タイトル

Particle agglutination method for poliovirus identification

著者

Minetaro Arita 1), Souji Masujima, Takaji Wakita 1), Hiroyuki Shimizu 1) 1) Department of Virology II, National Institute of Infectious Diseases

雑誌名

Journal of Visualized Experiments　50, 2011
http://www.jove.com/details.stp?id=2824 doi: 10.3791/2824.

内容

ポリオウイルス検査は、培養細胞およびPCRを用いた便検体からのウイルス分離・同定が行われているが、蔓延地域では検査体制をとりにくいこと、簡便な遺伝子検査（eg. LAMP）に利用できる保存配列がないこと、ウイルス株により抗原性の異なることが課題である。今回の手法は、①ポリオウイルス受容体と抗体のキメラ分子（APV-IgG2a）をゼラチン粒子に結合させ、ポリオウイルスを凝集反応で検出する方法である。また、②この凝集系に型特異的な抗血清を添加し凝集の競合阻害の有無によりポリオウイルスの型判別を行おうとするものである。

タイトル

Development of a novel rapid immunochromatographic test specific for the H5 influenza virus

著者

Eiji Miyagawa, Hiroyuki Kogaki, Yoshiaki Uchida, Nobuyuki Fujii, Takashi Shirakawa, Yoshihrio Sakoda 1), Hiroshi Kida 1)
1) Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

雑誌名

Journal of Virological Methods 173, 2011, p213-219

内容

Three anti-H5 influenza virus specific monoclonal antibody (mAb) clones,　IFH5-115, IFH5-136 and IFH5-26 were obtained by immunising of BALB/C mouse with inactivated A/duck/Hokkaido/Vac-1/04 (H5N1). These mAbs specifically recognized haemagglutinin (HA) of influenza H5 subtypes IFH5-115, IFH5-136 and IFH5-26 have no neutralizing activity for A/duck/Hokkaido/84/02 (H5N3) and A/PR/8/34 (H1N1). By epitope analysis using several deleted recombinant HA antigens of C- and N-terminal of A/duck/Hokkaido/Vac-1/04 (H5N1), from 46 to 60aa and from 312 to 322aa for IFH5-115, from 101 to 113 aa and from 268 to 273 aa for IFH5-136, and from 61 to 80 aa and from 290 to 300 aa for IFH5-26 were critical for immunological reactivities, respectively. The monoclonal antibodies recognize the conformational epitope in the loop regions between the receptor region and alfa-helix structure in hemagglutinin 1(HA1).
In order to detect influenza A virus H5 specifically, rapid immunochromatographic test kits based on the sandwich enzyme immunoassay (EIA) were constructed by the combination of the 3 mAbs; IFH5-26/IFH5-115, IFH5-136/IFH5-26 and IFH5-136/IFH5-115 as solid phase antibody/alkaline phosphates conjugated mAb. Each test detect the influenza H5, A/swan/Hokkaido/51/96 (H5N3), A/tern/South Africa/61 (H5N3), A/Hong Kong/483/91 (H5N1), A/duck/Yokohama/aq-10/03 (H5N1), A/Vietnam/1194/04 (H5N1), A/chicken/Yamaguchi/04/3/05 (H5N1), A/whooper swan/Mongolia/3/05 (H5N1) and A/whooper swan/Mongolia (H5N1) but not detected subtypes other than H5.