Name: antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae
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この研究は、チュニジア保健省とチュニジア高等教育科学研究省から資金提供を受けました。

本研究は、チュニスパスツール研究所の倫理委員会によって確立された倫理的側面に準拠して実施された。
1. ELISA前のステップ:前提条件と前処理
<ol><li>細菌株および増殖培地 注:本研究で使用されたモリキューテス種と壁に囲まれた細菌、およびそれらの増殖培地を 表1に示します。<ol><li>モリキューテス種の成長<ol><li>各種のグリセロールス...

<ol>
	<li>Razin, S., Yogev, D., Naot, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+and+pathogenicity+of+mycoplasmas.">Molecular biology and pathogenicity of mycoplasmas.</a> Microbiology and Molecular Biology Reviews. 62 (4), 1094-1156 (1998).</li><li>Chanock, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+infections+of+man.">Mycoplasma infections of man.</a> The New England Journal of Medicine. 273 (22), 1199-1206 (1965).</li><li>Braun, G. S., Wagner, K. S., Huttner, B. D., Schmid, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+pneumoniae:+Usual+suspect+and+unsecured+diagnosis+in+the+acute+setting.">Mycoplasma pneumoniae: Usual suspect and unsecured diagnosis in the acute setting.</a> The Journal of Emergency Medicine. 30 (4), 371-375 (2006).</li><li>Baseman, J. B., Cole, R. M., Krause, D. C., Leith, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+cytadhesion+of+Mycoplasma+pneumoniae.">Molecular basis for cytadhesion of Mycoplasma pneumoniae.</a> Journal of Bacteriology. 151, 1514-1522 (1982).</li><li>Waldo, R. H., Krause, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+stability,+and+function+of+cytadhesin+P1+and+accessory+protein+B/C+complex+of+Mycoplasma+pneumoniae.">Synthesis, stability, and function of cytadhesin P1 and accessory protein B/C complex of Mycoplasma pneumoniae.</a> Journal of Bacteriology. 188 (2), 569-575 (2006).</li><li>Waites, K. B., Balish, M. F., Atkinson, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+pathogenesis+and+detection+of+Mycoplasma+pneumoniae+infections.">New insights into the pathogenesis and detection of Mycoplasma pneumoniae infections.</a> Future Microbiology. 3 (6), 635-648 (2008).</li><li>Narita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+extrapulmonary+manifestations+of+Mycoplasma+pneumoniae+infection+with+special+reference+to+pneumonia.">Pathogenesis of extrapulmonary manifestations of Mycoplasma pneumoniae infection with special reference to pneumonia.</a> Journal of Infection and Chemotherapy. 16 (3), 162-169 (2010).</li><li>Kashyap, S., Sarkar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+pneumonia:+Clinical+features+and+management.">Mycoplasma pneumonia: Clinical features and management.</a> Lung India. 27 (2), 75-85 (2010).</li><li>Zhang, L., Zong, Z. Y., Liu, Y. B., Ye, H., Lv, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+versus+serology+for+diagnosing+Mycoplasma+pneumoniae+infection:+A+systematic+review+&amp;+meta-analysis.">PCR versus serology for diagnosing Mycoplasma pneumoniae infection: A systematic review &amp; meta-analysis.</a> Indian Journal of Medical Research. 134 (3), 270-280 (2011).</li><li>Saraya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+pneumoniae+infection:+Basics.">Mycoplasma pneumoniae infection: Basics.</a> Journal of General and Family Medicine. 18 (3), 118-125 (2017).</li><li>Jacobs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+diagnosis+of+Mycoplasma+pneumoniae+infections:+a+critical+review+of+current+procedures.">Serological diagnosis of Mycoplasma pneumoniae infections: a critical review of current procedures.</a> Clinical Infectious Diseases. 17, 79-82 (1993).</li><li>Kok, T. W., Marmion, B. P., Varkanis, G., Worswick, D. A., Martin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+diagnosis+of+Mycoplasma+pneumoniae+infection:+3.+Detection+of+IgM+antibodies+to+M.+pneumoniae+by+a+modified+indirect+haemagglutination+test.">Laboratory diagnosis of Mycoplasma pneumoniae infection: 3. Detection of IgM antibodies to M. pneumoniae by a modified indirect haemagglutination test.</a> Epidemiology and Infection. 103 (3), 613-623 (1989).</li><li>Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+pneumoniae+infections:+diagnosis+based+on+immunofluorescence+titer+of+IgG+and+IgM+antibodies.">Mycoplasma pneumoniae infections: diagnosis based on immunofluorescence titer of IgG and IgM antibodies.</a> Mayo Clinic Proceedings. 61 (10), 830-831 (1986).</li><li>Busolo, F., Tonin, E., Conventi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-linked+immunosorbent+assay+for+detection+of+Mycoplasma+pneumoniae+antibodies.">Enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antibodies.</a> Journal of Clinical Microbiology. 12 (1), 69-73 (1980).</li><li>Van Griethuysen, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+enzyme-linked+immunosorbent+assay+for+the+early+diagnosis+of+Mycoplasma+pneumoniae+infection.">Use of the enzyme-linked immunosorbent assay for the early diagnosis of Mycoplasma pneumoniae infection.</a> European Journal of Clinical Microbiology. 3 (2), 116-121 (1984).</li><li>Raisanen, S. M., Suni, J. I., Leinikki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+diagnosis+of+Mycoplasma+pneumoniae+infection+by+enzyme+immunoassay.">Serological diagnosis of Mycoplasma pneumoniae infection by enzyme immunoassay.</a> Journal of Clinical Pathology. 33 (9), 836-840 (1980).</li><li>Sasaki, T., Bonissol, C., Stoilikovic, B., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+cross-reactive+antibodies+to+mycoplasmas+in+human+sera+by+ELISA+and+immunoblotting.">Demonstration of cross-reactive antibodies to mycoplasmas in human sera by ELISA and immunoblotting.</a> Microbiology and Immunology. 31 (7), 639-648 (1987).</li><li>Brunner, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpectedly+high+frequency+of+antibody+to+Mycoplasma+pneumoniae+in+human+sera+as+measured+by+sensitive+techniques.">Unexpectedly high frequency of antibody to Mycoplasma pneumoniae in human sera as measured by sensitive techniques.</a> Journal of Infectious Diseases. 135 (4), 524-530 (1977).</li><li>Plackett, P., Marmion, B. P., Shaw, E. J., Lemcke, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+analysis+of+Mycoplasma+pneumoniae:+3.+Separation+and+chemical+identification+of+serological+active+lipids.">Immunochemical analysis of Mycoplasma pneumoniae: 3. Separation and chemical identification of serological active lipids.</a> Australian Journal of Experimental Biology and Medical Science. 47 (2), 171-195 (1969).</li><li>Ponka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occurrence+and+clinical+picture+of+serologically+verified+Mycoplasma+pneumoniae+infections+with+emphasis+on+central+nervous+system,+cardiac+and+joint+manifestations.">The occurrence and clinical picture of serologically verified Mycoplasma pneumoniae infections with emphasis on central nervous system, cardiac and joint manifestations.</a> Annals of Clinical Research. 11, 1-60 (1979).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72 (1-2), 248-254 (1976).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Towbin, H., Staehlin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets.+Procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications.</a> Proceedings of National Academy of Sciences. 76 (9), 4350-4354 (1979).</li><li>Ben Abdelmoumen, B., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-linked+immunosorbent+assay+for+detection+of+avian+mycoplasmas+in+culture.">An enzyme-linked immunosorbent assay for detection of avian mycoplasmas in culture.</a> Avian Diseases. 39, 85-93 (1995).</li><li>Fawcett, P. T., O'Brien, A. E., Doughty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adsorption+procedure+to+increase+the+specificity+of+enzyme-linked+immunosorbent+assays+for+lyme+disease+without+decreasing+sensitivity.">An adsorption procedure to increase the specificity of enzyme-linked immunosorbent assays for lyme disease without decreasing sensitivity.</a> Arthritis and Rheumatism. 32 (8), 1041-1044 (1989).</li><li>Nicolson, G. L., Nasralla, M. Y., Nicolson, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+and+treatment+of+mycoplasmal+infections.">The pathogenesis and treatment of mycoplasmal infections.</a> Antimicrobics and Infectious Disease Newsletter. 17 (11), 81-88 (1999).</li><li>Meyer, R. D., Clough, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extragenital+Mycoplasma+hominis+infections+in+adults:+emphasis+on+immunosuppression.">Extragenital Mycoplasma hominis infections in adults: emphasis on immunosuppression.</a> Clinical Infectious Diseases. 17, 243-249 (1993).</li><li>Pitcher, D. G., Nicholas, R. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+host+specificity:+Fact+or+fiction.">Mycoplasma host specificity: Fact or fiction.</a> Veterinary Journal. 170 (3), 300-306 (2005).</li><li>Lierz, M., Jansen, A., Hafez, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+lipofaciens+transmission+to+veterinarian.">Mycoplasma lipofaciens transmission to veterinarian.</a> Emerging Infectious Diseases. 14 (7), 1161-1163 (2008).</li><li>Baker, A. S., Ruoff, K. L., Madoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mycoplasma+species+from+a+patient+with+seal+finger.">Isolation of Mycoplasma species from a patient with seal finger.</a> Clinical Infectious Diseases. 27 (5), 1168-1170 (1998).</li><li>Slack, M., P, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+role+of+Haemophilus+influenzae+in+community-acquired+pneumonia.">A review of the role of Haemophilus influenzae in community-acquired pneumonia.</a> Pneumonia. 6 (1), 26-43 (2015).</li><li>Janira Avire, N., Whiley, H., Ross, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+Streptococcus+pyogenes:+public+health+risk+factors,+prevention+and+control.">A review of Streptococcus pyogenes: public health risk factors, prevention and control.</a> Pathogens. 10 (2), 248 (2021).</li><li>Steinitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+the+blocking+effect+of+Tween+20+and+bovine+serum+albumin+in+ELISA+microwells.">Quantitation of the blocking effect of Tween 20 and bovine serum albumin in ELISA microwells.</a> Analytical Biochemistry. 282 (2), 232-238 (2000).</li><li>Tully, J. G., Whitcomb, R. F., Clark, H. F., Williamson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+mycoplasmas:+cultivation+and+vertebrate+pathogenicity+of+a+new+spiroplasma.">Pathogenic mycoplasmas: cultivation and vertebrate pathogenicity of a new spiroplasma.</a> Science. 195 (4281), 892-894 (1977).</li><li>Frey, M. L., Hanson, R. P., Andrson, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+medium+for+the+isolation+of+avian+mycoplasmas.">A medium for the isolation of avian mycoplasmas.</a> American Journal of Veterinary Research. 29 (11), 2163-2171 (1968).</li><li>Bertani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+lysogenesis.+I.+The+mode+of+phage+liberation+by+lysogenic+Escherichia+coli.">Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli.</a> Journal of Bacteriology. 62 (3), 293-300 (1951).</li></ol>

この論文では、主に特定のスクリーニングを確実にするために開発された社内ELISAの一般的な説明を示します。 M. pneumoniae 感染症。ELISAアッセイ自体のプロトコルに関する詳細、およびいくつかの前処理および後処理ステップが提供されます。このアッセイの特異性は、吸着技術によって保証されます。この手順は、ヒトおよび鳥類の診断用に開発されたELISA試験で以前に説明されてい?...

マイコプラズマは 、最も小さく、最も単純な既知の原核生物の一つです。それらは主に細胞壁構造の欠如によって他の細菌と区別される。したがって、 マイコプラズマは モリキューテス1という名前の別のクラスに分類されました。細胞壁欠損症は、いくつかの抗菌剤に対してこれらの微生物に対する固有の耐性を与え、それらの多型の主な原因です。 マイコプラズマは ゲノムが小さく、サイズが小さく、代謝および生合成能力が制限され、寄生および腐生の性質を説明しています1。
マイコプラズマ・ニューモニエは、人間に感染するマイコプラズマの1つであり、最も毒性が高いと考えられています2。M.肺炎は上気道にコロニーを形成し、小児および若年成人に非定型肺炎を引き起こします。肺炎球菌感染によって引き起こされる臨床徴候はインフルエンザのようなもので、頭痛、発熱、咳3があります。宿主細胞に対するM. pneumoniaeの細胞付着は、P1主要な接着およびいくつかの付属品タンパク質を含む付着オルガネラによって媒介される4,5。気道粘膜へのM.肺炎の密接な付着に起因する局所的な炎症および宿主免疫系の刺激のために、より多くの臨床症状が発生する可能性があります6。肺炎は肺炎菌感染の特徴ですが、この細菌による感染は、中枢神経系、心臓、皮膚、関節などのさまざまな解剖学的部位における広範囲の非肺症状の原因にもなる可能性があることが明らかになっています7。
すべてのマイコプラズマ種に関して、M.肺炎の診断は困難です。マイコプラズマ症を誘発する臨床徴候はほとんど不明であり、特徴的ではありません8。肺炎球菌感染症を臨床症状と症状のみに頼って診断することは非常に難しいため、臨床検査は特に興味深いものです9。培養によるM.肺炎コロニーの検出は、適切な診断のためのゴールドスタンダードの方法です。しかし、慎重な成長要件と決定的な結果の提供に必要な長い時間(1〜2週間)は培養を複雑にし、したがってそれが日常的な診断に使用されることはめったにないことを意味します10。核酸増幅技術は速度と効率の点で検証されましたが、コストが比較的高く、一部の医療施設では利用できないため、これらの分子技術は第一選択の診断テストとは見なされません。市販のPCR検査が肺炎球菌感染症の診断に広く使用されているのは事実ですが、それでも血清学に取って代わることはできません。また、偽陰性と偽陽性の両方の結果が頻繁に発生するため、PCR9の使用が制限されています。日常的に、血清学は、肺炎球菌感染症の診断のために実験室で最も実践されています。冷赤血球凝集素、補体結合試験11、間接赤血球凝集試験12、免疫蛍光13、および1980年代初頭にマイコプラズマ血清学に最初に適用されたELISAの技術など、いくつかの血清学的アプローチが数十年にわたって報告されています14,15,16。肺炎球菌感染症のELISA血清診断を行う際に遭遇する主要な問題の1つは交差反応であり、これは技術の特異性をかなり低下させる。M.肺炎抗原によるヒト血清の非特異的吸着は以前に報告されています。実際、ヒト血清中のELISAによって検出された抗体の多くは、一部の細菌18,19および一部の動物およびヒト組織とのM.肺炎の共通性のために、マイコプラズマ抗原17に常に結合しているとは限りません20。
実験室で実施された従来のELISA検査で観察された高いバックグラウンド測定値のために、結果の解釈はしばしば複雑であり、したがって適切な肺炎球菌診断の提供は困難な課題でした。この問題に直面しながら、我々は、M. pneumoniae抗原と試験する抗体との非特異的反応を除去することにより、M. pneumoniae ELISAを改善することを選択しました。この目的のために、吸着技術を用いて非特異的肺炎球菌抗原の選択的枯渇に取り組みました。実際、抗原捕捉ELISAの主な目的は、ヒト血清サンプル中の肺炎球菌免疫グロブリン(Ig)Gを特異的に検出することです。このELISAの概念は、主にヒト血清サンプルを追加する前に、肺炎球菌特異的抗原を選択的に捕捉することで構成されています。この選択性は、実験室でウサギで産生されたM.肺炎粗抗原をM.肺炎ポリクローナル抗血清とインキュベートし、モリキューテスクラスに属するかどうかにかかわらず、M.肺炎と抗原を共有する異種細菌のパネルに対する吸着によって種特異的にすることによって保証されます。 種および/または気道にコロニーを形成することが知られている。吸着手順を3回繰り返し、交差反応性を排除する効率をイムノブロッティングで試験した。開発されたELISAアッセイは、サンドイッチELISAと間接ELISAの組み合わせです。簡単に説明すると、ELISAプレートのウェルは、最初にM.ニューモニエに特異的なポリクローナル抗血清でコーティングされる。次いで、M. pneumoniae抗原が添加され、抗血清と試験対象の血清サンプル中に存在する抗体との間に捕捉される。形成された免疫学的複合体は、二次酵素結合抗体(ペルオキシダーゼ結合IgG)によって検出される。反応は発色基質の添加によって視覚化され、吸光度は分光光度法で測定されます。この社内ELISAを図1に模式的に示します。自家製のELISAは、肺炎球菌感染を特異的に検出するのに効率的であることが証明され、現在、日常的な診断活動で最も実践されている検査の1つです。

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			<td>4-chloro-1-naphtol</td>
			<td>Sigma-Aldrich</td>
			<td>C6788-50 TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>BD</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-50 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate bicarbonate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>C3041-50 Cap</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C7078-1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CMRL1066&#160;</td>
			<td>VWR</td>
			<td>P0058-N1L</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco PPLO Broth</td>
			<td>BD</td>
			<td>255420</td>
			<td>Frey media</td>
		</tr>
		<tr>
			<td>ELISA plate-Reader</td>
			<td>MULTISKAN GO, Thermo Scientific</td>
			<td>Ref: 51119200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Capricorn Scientific</td>
			<td>FBS-12A</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat peroxidase-conjugated anti-human IgG</td>
			<td>Abcam</td>
			<td>ab6759</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat peroxidase-conjugated anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>656120</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid 37%</td>
			<td>Prolab</td>
			<td>2025.290</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2), solution 30%</td>
			<td>Scharlau</td>
			<td>HI01361000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-arginin</td>
			<td>Sigma-Aldrich</td>
			<td>A5006-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Prepared in Pasteur Institute of Tunis</td>
			<td></td>
			<td>Provided by the laboratory of bacteriology of the Pasteur Institute of Tunis</td>
		</tr>
		<tr>
			<td>Mycoplasma pneumoniae polyclonal antiserum produced in rabbit</td>
			<td>Produced in Pasteur Institute of Tunis</td>
			<td></td>
			<td>Serum was produced by rabbit immunization at the Pasteur Institute of Tunis</td>
		</tr>
		<tr>
			<td>Nicotinamide adenine dinucleotide</td>
			<td>Sigma-Aldrich</td>
			<td>N7004-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Maxisorp flat-bottom 96-well microtiter plate&#160;</td>
			<td>Invitrogen</td>
			<td>44-2404-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium (1 MIU)</td>
			<td>PANPHARMA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>fluka chemika</td>
			<td>77660</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>MP Biomedicals</td>
			<td>902887</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S6297-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S7795-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Novachim</td>
			<td>PS02805</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid (H2SO4) 95-97%</td>
			<td>Merck</td>
			<td>1007311011</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal</td>
			<td>USB</td>
			<td>22215</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB substrate (3,3&#8217;, 5,5&#8217; TetraMethyl-Benzidine solution)</td>
			<td>Abcam</td>
			<td>ab142042</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T6791-1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>1379-500 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract, powder, Ultrapure</td>
			<td>Thermo Scientific&#160;</td>
			<td>J23547&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae

マイコプラズマ・ニューモニエ は細胞壁欠損原核生物であり、主にヒトの気道にコロニーを形成し、流行していることが知られており、年長の子供や若年成人では6年ごとに流行のピークがあります。 M.肺炎 の診断は、病原体の潔癖な性質と無症候性の運搬の可能性があるため、困難です。患者の血清サンプル中の抗体滴定に基づく 肺炎 菌感染症の臨床検査は、依然として最も実践されている方法である。 M. pneumoniaeに対するポリクローナル血清の使用による免疫学的交差反応性の潜在的な問題のために、血清学的診断の特異性を改善するために抗原捕捉酵素結合免疫吸着アッセイ(ELISA)が開発されました。ELISAプレートは、 M. pneumoniae ポリクローナル抗体でコーティングされ、ウサギで飼育され、 M. pneumoniae 種と抗原を共有する、および/または気道にコロニーを形成することが知られている異種細菌のパネルに対する吸着後に特異的になります。反応した M. pneumoniae 相同抗原は、次いで、血清サンプル中のそれらの対応する抗体によって特異的に認識される。抗原捕捉ELISAが受ける物理化学的パラメータのさらなる最適化は、非常に特異的で、感度が高く、再現性の高いELISAにつながりました。

異種細菌に対する非吸着ポリクローナルマイコプラズマ・ニューモニエ抗血清のイムノブロッティング活性 イムノブロッティングの結果(図2)で示されているように交差反応性は実際に存在し、ポジティブコントロール(レーン1)と比較して、肺炎球菌抗原の一部はスクリーニングされた細菌と共有されています。こ?...

Watch this Scientific Journal Video about Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae at JoVE.com

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae

マイコプラズマ・ニューモニエ感染症では、血清学的検査は良好な結果を生み出すことができますが、免疫学的交差反応のために特異性は低くなります。この論文に記載されている社内抗原捕捉ELISAは、高い種特異性を保証し、肺炎球菌の正確な診断のための信頼できるスクリーニング検査であることが示されています。

マイコプラズマ・ニューモニエの特異的検出のための抗原捕捉酵素結合免疫吸着アッセイ(英語)

Mycoplasma pneumoniae is a cell wall-deficient prokaryote, mainly known to colonize the human respiratory tract and to be endemic, with epidemic peaks ...

すべてのマイコプラズマ患者に関しては、マイコプラズマ肺炎の診断は交差反応のために困難です。我々が提案するELISAは、吸着技術を用いた非特異的肺炎マイコプラズム抗原の選択的枯渇に基づいている。このビデオで説明されている社内抗原捕捉ELISAは、高い広い特異性を保証します。そして実際には、肺炎マイコモニエ感染症を正確に診断するための信頼できるスクリーニング検査であることが証明されています。ELISA前のステップを実演するのは、博士課程の学生であるイメン・クニバです。そして、ELISAのステップは、私たちの研究室の技術者であるナディーン・カドラウィです。抗原吸着手順を開始するには、12個の異種細菌抗原を微小遠心チューブに引き込み、吸着する抗血清の半分に相当するタンパク質濃度を調整します。次に、抗原混合物を摂氏4度で14, 000 gで10分間遠心分離し、ペレットを回収します。次に、精製されたポリクローナル抗マイコプラズマ肺炎IgGでペレットを再懸濁し、ゆっくりと攪拌しながら摂氏37度で2時間インキュベートします。インキュベーションの終わりに、懸濁液を摂氏4度で10分間14, 000Gで再び遠心分離し、特定のポリクローナルマイコプラズマ肺炎抗血清に対応する上清を回収する。96ウェルELISAプレートを捕捉抗体でコーティングするには、抗体を1ミリリットルあたり10マイクログラムに希釈し、pH 9.6で0.1モルの炭酸水素塩バッファーで希釈します。次に、100マイクロリットルの希釈抗体をマイクロプレートの各ウェルに加え、摂氏4度で一晩インキュベートします。翌日、コーティング溶液を取り出し、プレートを洗浄バッファーで5回洗浄します。次に、各ウェルに100マイクロリットルのブロッキング溶液を加えて、コーティングされたウェルの未結合表面をブロックします。プレートを室温で1時間インキュベートした後、ブロッキング溶液をピペットで除去し、前述のようにプレートを洗浄します。同量のマイコプラズマ・ニューモニエ全タンパク質をウェルあたり10ナノグラムの濃度で添加し、抗原抗体反応を室温で2時間起こします。インキュベーション後、余分な抗原を取り除き、実証されているようにプレートを洗浄します。次に、ヒト血清試験サンプルを希釈し、陽性および陰性の血清対照を参照します。次に、100マイクロリットルの希釈した試験サンプルとコントロールを適切なウェルと複製に追加します。抗原も血清も含まないウェルをブランクとしてマークします。プレートを室温で90分間インキュベートした後、血清溶液を取り出し、プレートを洗浄バッファーで5回洗浄します。検出抗体を各ウェルに100マイクロリットルの適切な希釈酵素結合体をピペットし、暗所で摂氏37度で1時間インキュベートする。結合していない検出体を除去し、プレートを洗浄した後、100マイクロリットルのTMBクロモージェン溶液を加えて、抗原抗体結合を可視化します。プレートを室温で30分間発達させます。その後、100マイクロリットルの7.5%硫酸を加えて反応を停止します。最後に、マイクロプレートリーダーを使用して450ナノメートルの吸光度を読み取り、陽性指数の計算に基づいて結果を並べ替えます。マイコプラズマ・ニューモニエ非吸着ポリクローナル抗血清と異種細菌抗原との間のイムノブロット分析では、交差反応性が明らかになりましたが、強度はさまざまでした。例えば、マイコプラズマ・ガリセプティカムとマイコプラズマ模倣抗原は、トリ類マイコプラズマであるにもかかわらず、最も強い反応性を示しました。対照的に、尿素プラズマ尿素溶解の2つのヒト臨床分離株のどちらも反応を示さなかった。しかし、残りのヒト性器マイコプラズマ種の抗原および他の細菌抗原は、抗血清とかなりの交差反応をもたらした。イムノブロット分析により、異種細菌抗原に対するマイコプラズマ・ニューモニエ・ポリクローナル抗血清吸着の有効性が確認されました。吸着手順により、すべての交差反応が排除され、マイコプラズマ肺炎抗血清がその後のすべての血清学的およびイムノブロッティング試験に特異的になりました。さらに、本研究で開発した抗原捕捉ELISAを用いて試験したヒト血清セットは、良好な特異性を有するマイコプラズマ・ニューモニエIgGに対して陽性であることが証明された。このELISAの特異性は、この重要なステップを注意深く実行し、少なくとも2回または3回繰り返すことが原始的であるため、主に吸着技術によって保証されます。この技術の原理は、他のマイコプラズマ種または他の細菌一般の診断に適用することができる。しかしながら、吸着のための細菌抗原の選択は無関係かつ適切であるべきである。

antigen-capture-enzyme-linked-immunosorbent-assay-for-specific

Research

JoVE Journal

Biology

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

タンパク質のユビキチン化の検出

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

生物学

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME（免疫磁気電気化学的にリンクされた酵素）マイコトキシンの検出方法

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

遺伝的関連の機能的基準をテストするための対立遺伝子特異的遺伝子発現アッセイ

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

初期段階のマウス胚を用いて組織特異的遺伝子のためのクロマチン免疫沈降アッセイ

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

マイコプラズマの最適化されたPCRベースの検出

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

培養細胞における細胞内および細胞外アスコルビン酸の定量のための迅速かつ特異的マイクロプレートアッセイ

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

マラリア蚊。送信のためのマルチ検出分析

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

蛍光標識ナノ粒子を用いたマウス、ヒトおよびブタ起源のライブ細胞の細胞内遺伝子発現の検出

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

部位特異的DNA切断のための並列高スループット単一分子運動アッセイ

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.