Finanzielle Unterstützung für Dr. Thornton von Pfizer Limited wird dankbar anerkannt.

1. Sammlung, Lagerung und Vorbereitung von Serum-und bronchoalveolären Lavage <ol><li> Serum, entnommen aus unbehandelten Blutproben, indem sie die Blutgerinnung bei 4 ° C, und speichern Serum als Aliquots bei -20 ° C erwärmen lassen. Bronchoalveolären Lavage (BAL) sollte auch als Aliquots bei -20 ° C gelagert werden Hinweis: Die Lagerung von Serum und BAL als Aliquots das Potenzial für den Abbau der Ziel-Antigen, das durch wiederholtes Einfrieren und Auftauen der Proben während einer Wiederholung des Tests begrenzt. </li><li> Tauwetter Proben und mischen Sie die Serum und BAL-Proben gründlich durch Vortexen und Zentrifugation für 1 min bei 14.000 Umdrehungen pro Minute. </li><li> Für die Routineprüfung der menschlichen Serumproben, verdünnt das Serum 1:1 (v / v) mit Gewebekulturmedium (TCM). TCM besteht aus RPMI-1640-Medium, 10% (v / v) fötalem Kälberserum, 1% (v / v) von 200 mM L-Glutamin-Lösung, und Natriumazid (0,02% w / v) als Konservierungsmittel. Das Medium wird im Voraus vorbereitet und können bei 4 ° C für mehrere Monate gelagert werden. Tragen Sie 100 ul der DILUTed Serum auf die LFD. </li><li> Für menschliche BAL-Flüssigkeiten, und für BAL-Flüssigkeiten aus Tiermodellen, gelten 100 ul Probe ordentlich auf die LFD, ohne Vorbehandlung. </li><li> Für Serum von Tiermodellen, verdünnte Serum 1:2 (v / v) mit Phosphat-gepufferter Salzlösung, enthaltend 4% (w / v) EDTA, Wärme für 3 min in einem kochenden Wasserbad, Zentrifuge für 5 min bei 14.000 rpm und Einsatzes von 100 ul des reinen Überstands an die LFD-Gerät. </li></ol> 2. Anwendung von Serum und BAL auf die Lateral-Flow Geräte- <ol><li> Lagern Sie die Lateral-Flow-Geräten bei Raumtemperatur (23 ° C). Bei dieser Temperatur sind Geräte für 12 Monate stabil. Entfernen Sie die Geräte aus den Beuteln und Ort auf einer ebenen Fläche. </li><li> Mit einer sterilen Pipettenspitze, gelten 100 ul vorbehandelten Serum oder ordentlich BAL-Probe zur Freisetzung Port des Geräts. </li><li> Lassen Sie die Assay für 15 Minuten bei Raumtemperatur, zu welchem ​​Zeitpunkt die Ergebnisse der Prüfungen aufzuzeichnen ausgeführt werden soll. Hinweis: Innerhalb von Sekunden wird die Flüssigkeit an sich seinde durch Kapillarwirkung entlang der Nitrocellulose-Membran im Beobachtungsfenster zu migrieren. </li></ol> 3. Aufnahme und Interpretation LFD Ergebnisse <ol><li> Die LFD besteht aus einem internen Steuerleitung (angedeutet durch den Buchstaben C auf dem Kunststoff-Gehäuse) und eine Testlinie (gekennzeichnet mit dem Buchstaben T). Die Steuerung sollte in jedem Fall unabhängig von Aspergillus-Antigen werden im Serum oder BAL-Probe. Dies zeigt, dass der Test korrekt ausgeführt werden. </li><li> Wenn die Aspergillus-Antigen im Serum oder BAL-Probe, wird die Testlinie auch innerhalb 15 Minuten der Probe Anwendung angezeigt. Da die Intensität der Testlinie ist proportional zu der Menge von Aspergillus-Antigen in der Probe vorhanden, die Testlinie kann schwach positiv (+) angezeigt wird, eine moderate positiv (+ +) oder als stark positiv (+ + +) . Jedoch eine beliebige positive Testleitung, unabhängig von Intensität, deutet dies auf der Aspergillus-Antigen in der Probe. In the Abwesenheit von Aspergillus-Antigen, wird keine Testlinie erscheinen, und das Ergebnis wird als negativ aufgenommen (-). </li></ol> 4. Repräsentative Ergebnisse Repräsentative Beispiele für negative, schwach positive, mäßig positive und stark positive Ergebnisse mit LFD BAL und Serum-Proben sind in Abbildung 1 dargestellt. Ergebnisse der Antigen-positiven Tests LFD (schwache und starke) oder negativ LFD Tests mit BAL-Flüssigkeiten mit akuter myeloischer Leukämie (AML) nach Anspruch EORTC / MSG diagnostischen Kriterien diagnostiziert werden in Tabelle 1 gezeigt. In dieser Tabelle sind die entsprechenden klinischen und mykologischen (Galaktomannan und Kultur) Daten und Ergebnisse einer Aspergillus-spezifischen PCR-Test in St. Bartholomews Krankenhaus 8 entwickelt, für jeden Patienten. Die Diagnose der Krankheit wurde am Wirtsfaktoren (Neutropenie, verlängerter Einsatz von Kortikosteroiden, Behandlung mit anderen anerkannten T-Zell-immunos Basisuppressants), klinische Kriterien und GM Positivität für BAL (hier als GM-Index-Wert&gt; 0,8 definiert). Unter den 2002 EORTC / MSG-Richtlinien 10, Patienten, 12, 13 und 16 wurden mit &quot;möglich&quot; IA auf der Basis von Host-Faktoren und klinischen Kriterien oder GM Positivität diagnostiziert. Nach den revidierten (2008) EORTC / MSG-Richtlinien 2, Wirtsfaktoren und GM Positivität allein oder Host-Faktoren und klinischen Kriterien allein würde nicht darauf hin, &quot;möglich&quot; invasiven Pilzinfektion, es sei denn durch Belege aus klinischen Daten und Mykologie bzw. begleitet. Patient 6 wurde mit &quot;wahrscheinlich&quot; IA unter beiden Richtlinien 2002 und 2008 wegen der Wirtsfaktoren, größere und kleinere klinische Merkmale und GM Positivität diagnostiziert. Beachten Sie, dass, während weder die LFD noch PCR-Assays zur Zeit in EORTC / MSG-Richtlinien enthalten sind, gibt es starke Übereinstimmung zwischen den beiden Assays und der kommerziellen Galaktomannan Test auf das Vorhandensein von Aspergillus-Antigen und Nukleinsäure in der BAL-Probe<html> s der Patienten 6 und 12. Zusätzliche Ergebnisse von Studien, die die Wirksamkeit der LFD und PCR-Test bei der Diagnose von IPA kann in Johnson et al 8 zu finden. <img alt="1" src="/files/ftp_upload/3721/3721fig1.jpg" /> Abbildung 1. Negativ (Steuerleitung nur) und positiven (Kontroll-und Testlinie) Ergebnisse der Tests mit LFD Serum und BAL. Die Intensitäten der Testlinie Reaktionen sind proportional zu den Konzentrationen der Ziel-Antigen im Serum und BAL-Proben. Reaktionen typischerweise im Bereich von schwach (+) durch moderate (+ +) bis stark (+ + +). Unabhängig von Testlinie Intensität würden alle drei Serum-positiven Reaktionen zeigen Lungenaspergillosen Krankheit aufgrund der Anwesenheit von zirkulierenden Aspergillus-Antigen im Blut. Positiv BAL Reaktionen (schwach, mittel oder stark) würde anzeigen, Keimen der Sporen und Entwicklung von potentiell pathogenen Hyphen in der Lunge. pdflinebreak &quot;&gt; <table border="1"><tbody><tr><td> Patient nicht. </td><td> PatiHNO-Informationen </td><td> Klinische Kriterien 1 </td><td> BAL Kultur 2 </td><td> GM EIA Index 3 </td><td> EORTC / MSG (2002) 4 </td><td> EORTC / MSG (2008) 5 </td><td> Aspergillus PCR-Ergebnis </td><td> LFD Ergebnis </td></tr><tr><td> 6 </td><td> - </td><td> Große CT Zeichen (Knötchen und Halo) und eine kleinere </td><td> Negativ </td><td> 0,9 (positiv) </td><td> Wahrscheinlich </td><td> Wahrscheinlich </td><td> Positiv </td><td> Schwache positive (+) </td></tr><tr><td> 12 </td><td> Aus Mangel an früheren Pilzinfektion </td><td> Kein großer, ein minderjähriges </td><td> Candida glabrata </td><td> 6,43 (stark positiv) </td><td> Möglich </td><td> Keiner </td><td> Positiv </td><td> Starke positive (+ + +) </td></tr><tr><td> 13 </td><td> Koagulase-negative Staphylokokkenylococcus und E. coli im Blut </td><td> Keine große, zwei kleinere </td><td> Negativ </td><td> 0,25 (negativ) </td><td> Möglich </td><td> Keiner </td><td> Negativ </td><td> Negative (-) </td></tr><tr><td> 16 </td><td> Brust-Infektion </td><td> 3 Minor Kriterien einschließlich neuer infiltrieren und Plural Erguss </td><td> Negativ </td><td> 0,16 (negativ) </td><td> Möglich </td><td> Keiner </td><td> Negativ </td><td> Negative (-) </td></tr></tbody></table> Ein Knötchen oder Halos auf einer Computertomographie ist suggestive der Pilzinfektion 2 Candida glabrata in der BAL würde als Verunreinigung angesehen werden, da es die zweithäufigste Hefe als Teil der normalen menschlichen Flora 9 isoliert ist 3 Ein Index von&gt; 0,8 in der GM EIA Test der BAL ist bezeichnend für Aspergillus-Infektion 4 auf den 2002 EORTC / MSG diagnostischen Kriterien für &#39;Basierend possible &quot;,&quot; wahrscheinlich &quot;oder&quot; bewiesen &quot;invasive Pilzerkrankung 10 5 auf den überarbeiteten (2008) EORTC / MSG diagnostischen Kriterien für &quot;möglich&quot;, &quot;wahrscheinlich&quot; oder &quot;bewiesen&quot; invasive Pilzerkrankung 2 Basierend Tabelle 1. Die Ergebnisse der Tests von LFD BAL-Proben von akuter myeloischer Leukämie Patienten mit wahrscheinlicher IPA und Kontrolle von AML-Patienten (keine Anzeichen einer Infektion) und EORTC / MSG Diagnose einer Infektion.

<ol>
	<li>Thornton, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+invasive+aspergillosis.">Detection of invasive aspergillosis.</a> Adv. Appl. Microbiol. 70, 187-216 (2010).</li><li>De Pauw, B., Walsh, T. J., Donnelly, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+definitions+of+invasive+fungal+disease+from+the+European+Organization+for+Research+and+Treatment+of+Cancer/Invasive+Fungal+Infections+Cooperative+Group+and+the+National+Institute+of+Allergy+and+Infectious+Diseases+Mycology+Study+Group+(EORTC/MSG)+concensus+group.">Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) concensus group.</a> Clin. Infect. Dis. 46, 1813-1821 (2008).</li><li>Thornton, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+emerging+human+pathogen+Pseudallescheria+boydii+by+using+highly+specific+monoclonal+antibodies.">Tracking the emerging human pathogen Pseudallescheria boydii by using highly specific monoclonal antibodies.</a> Clin. Vacc. Immunol. 16, 756-764 (2009).</li><li>Pickering, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+(1+->+3)-&#946;-D-glucan+assay+for+diagnosis+of+invasive+fungal+infections.">Evaluation of a (1 -> 3)-&#946;-D-glucan assay for diagnosis of invasive fungal infections.</a> J. Clin. Microbiol. 43, 5957-5962 (2005).</li><li>Thornton, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+immunochromatographic+lateral-flow+device+for+rapid+serodiagnosis+of+invasive+aspergillosis.">Development of an immunochromatographic lateral-flow device for rapid serodiagnosis of invasive aspergillosis.</a> Clin. Vacc. Immunol. 15, 1095-1105 (2008).</li><li>Verweij, P. E., Mennink-Kersten, M. A. S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Issues+with+galactomannan+testing.">Issues with galactomannan testing.</a> Med. Mycol. 44, 179-183 (2006).</li><li>Wiederhold, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+lateral+flow+technology+and+galactomannan+and+(1+->+3)-&#946;-D-glucan+assays+for+detection+of+invasive+pulmonary+aspergillosis.">Comparison of lateral flow technology and galactomannan and (1 -> 3)-&#946;-D-glucan assays for detection of invasive pulmonary aspergillosis.</a> Clin. Vacc. Immunol. 16, 1844-1846 (2009).</li><li>Johnson, G. L., Bibby, D., Bustin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Aspergillus+in+broncho-alveolar+lavage+fluid+using+two+biological+assays;+evidence+of+active+infection.">Detection of Aspergillus in broncho-alveolar lavage fluid using two biological assays; evidence of active infection.</a> Mycoses. 54, 130-131 (2011).</li><li>Malani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+glabrata+fungemia:+experience+in+a+tertiary+care+centre.">Candida glabrata fungemia: experience in a tertiary care centre.</a> Clin. Infect. Dis. 41, 975-981 (2005).</li><li>Ascioglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+opportunistic+invasive+fungal+infection+in+immunocompromised+patients+with+cancer+and+hematopoietic+stem+cell+transplants:+an+international+concensus.">Defining opportunistic invasive fungal infection in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international concensus.</a> Clin. Infect. Dis. 34, 71-74 (2002).</li><li>Denning, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+diagnosis+of+invasive+aspergillosis.">Early diagnosis of invasive aspergillosis.</a> Lancet. 355, 423-424 (2000).</li><li>Greene, R., Shibuya, K., Ando, T., Latge, J. -. P., Steinbach, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aspergillus fumigatus and Aspergillosis. , 353-363 (2009).</li><li>Klont, R. R., Mennink-Kersten, M. A. S. H., Verweij, 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=Utility+of+Aspergillus+antigen+detection+in+specimens+other+than+serum+samples.">Utility of Aspergillus antigen detection in specimens other than serum samples.</a> Clin. Infect. Dis. 39, 1467-1474 (2004).</li><li>Iweala, O. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+diagnostic+tests:+an+overview.">HIV diagnostic tests: an overview.</a> Contraception. 70, 141-147 (2004).</li><li>Ketema, F., Zeh, C., Edelman, 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=Assessment+of+the+performance+of+a+rapid,+lateral+flow+assay+for+the+detection+of+antibodies+to+HIV.">Assessment of the performance of a rapid, lateral flow assay for the detection of antibodies to HIV.</a> J. Acquir. Immune Defic. Syndr. 27, 63-70 (2001).</li><li>Moody, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+diagnostic+tests+for+malaria+parasites.">Rapid diagnostic tests for malaria parasites.</a> Clin. Microbiol. Rev. 15, 66-78 (2002).</li><li>Parkash, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+a+lateral+flow+test+for+the+detection+of+leprosy+patients+in+India.">Performance of a lateral flow test for the detection of leprosy patients in India.</a> J. Med. Microbiol. 57, 130-132 (2008).</li><li>Sharma, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+lateral-flow+Clostridium+botulinum+neurotoxin+detection+kits+for+food+analysis.">Evaluation of lateral-flow Clostridium botulinum neurotoxin detection kits for food analysis.</a> Appl. Environ. Microbiol. 71, 3935-3941 (2005).</li><li>Smits, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral-flow+assay+for+rapid+serodiagnosis+of+human+leptospirosis.">Lateral-flow assay for rapid serodiagnosis of human leptospirosis.</a> Clin. Diagn. Lab. Immunol. 8, 166-169 (2001).</li><li>Faulstich, K., Wong, R., Tse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lateral Flow Immunoassay. , 157-185 (2009).</li></ol>

Wir haben nichts zu offenbaren.

Definitive Identifizierung von IPA können nur dann wirklich durch Isolierung der Erreger aus Biopsie-Proben erreicht werden, sondern Wiederherstellung geeigneter Proben ist häufig nicht in sehr kranken Patienten möglich und Aspergillus ist selten wiederherstellbar aus dem Blut. Während die großen Fortschritte in der Anwendung von Computertomographie Abtasten der Brust in IPA Diagnose gemacht worden sind, sind Eigenschaften, die suggestive der pulmonalen IPA wie der &quot;Halo&quot; oder &quot;Luft-Halbmond&quot;-Schilder sind entweder flüchtig oder kann, um die Atmung Artefakten zugeschrieben werden oder andere Pilzinfektionen 11,12. Solche Daten sind deshalb mit serologischen Techniken, die zur Unterzeichnung Moleküle (GM und β-Glucan) aus Pilzen, die im Serum des Patienten im Umlauf sind oder, die in BAL-Flüssigkeiten, Sputum oder Urin-Proben ermittelt werden, 13 sind ergänzt. Während diese Tests zufriedenstellend Empfindlichkeit zeigen, fehlt ihnen ausreichende Spezifität oder leiden an Störungen unter bestimmten Bedingungen 1,6 </sup&gt;. Die LFD-Test für den IPA-Erkennung hier vorgestellten ermöglicht das &quot;Point-of-Care&quot; Diagnostik von IPA und Exploits Technologie, die verwendet wurde, um in Tests für den Nachweis von Viren, Bakterien, Parasiten und Giftstoffe 14-19 Laufenden und, am bekanntesten, für die Schwangerschaftstests zuerst von Unipath 1988 eingeführt. In der hier beschriebenen Aspergillus LFD wird die Aspergillus-spezifischen MAb JF5 einer Einfangzone (die Testlinie) auf einer porösen Nitrozellulosemembran immobilisiert. Anti-Maus-Immunglobulin immobilisiert auf die Membran in einer gesonderten Zone diente als eine interne Kontrolle (control line). Bei Zugabe von Serum oder BAL-Flüssigkeit zu der Öffnung für die Freisetzung bindet MAb JF5-Gold-Komplexe in der Freigabestellung Kontaktstelle mit der Ziel-Antigen und der Komplex dann entlang der porösen Membran durch Kapillarwirkung. MAb JF5 in der Abfangzone immobilisiert bindet an die JF5-Gold-Antigen-Komplexes, was zu einer roten Testlinie. Nicht gebundenes JF5-GoldKonjugat bindet an die interne Kontrolle darauf hinweist, dass der Test korrekt ausgeführt werden. Dies führt zu einer rote Kontrolllinie. Der Test ist schnell, wobei nur 15 Minuten zu führen, ist billig im Vergleich zu Serum und BAL Tests an GM und β-Glucan-Erkennung basiert und erfordert keine teure Ausrüstung oder umfangreiche Laboreinrichtungen zu laufen. Darüber hinaus bedeutet MAb JF5 nicht mit den Drogen oder Verunreinigungen, die gezeigt haben, um falsch-positive Reaktion in der GM-und β-Glucan-Tests 1,4,6 verursachen kreuzreagieren. Ein weiterer großer Vorteil gegenüber aktuellen diagnostischen Tests ist die Fähigkeit, LFD Aktivität, die indikativ für invasive Wachstum von Aspergillus-Spezies ist zu erkennen. Ein entscheidender Schritt bei der LFD Verfahren ist die Notwendigkeit zum Lesen der Ergebnisse von 15 min nach Anwendung des Serum oder BAL auf die Vorrichtung. Der Test sollte nicht länger als 15 min vor der Aufnahme die Ergebnisse gelassen werden, da dies zu Verzerrungen führen könnte interpretatiauf. Eine schwache Reaktion wird nicht durch die Verlängerung der Inkubationszeit verbessert werden. Wärmebehandlung von Serum aus Tiermodellen der Infektion wurde vor dem Test die Empfindlichkeit zu verbessern. Eine Einschränkung des Tests ist, dass es qualitative ist, und setzt auf die Betreiber, um eine subjektive Einschätzung der Positivität zu machen. Die Intensität der Testlinie variiert je nach den Antigen Inhalt Serum und BAL-Proben (1). Jedoch zeigt eine positive Reaktion (bestimmt durch Vergleich mit bekannten Negative) das Vorhandensein von Aspergillus Antigen und daher Infektion. Um die Subjektivität der LFD-Assays zu begrenzen, sind Handheld-Geräte verfügbar, die es erlauben die Quantifizierung der LFD Testlinie Intensitäten und ermöglichen die Festlegung von Schwellenwerten für Antigennachweis 20. Zukünftige Entwicklungen des LFD sind seine Kommerzialisierung und die Entwicklung eines Multiplex-LFD, die simultane Detektion von anderen invasiven Pilzerreger erlaubtmit hochspezifischen MAbs 3.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Aspergillus LFD </td><td> University of Exeter </td><td></td><td> Frei ab dem entsprechenden Autor auf Anfrage </td></tr><tr><td> RPMI-1640 </td><td> Sigma </td><td> R0883 </td><td></td></tr><tr><td> L-Glutamin </td><td> Sigma </td><td> G7513 </td><td></td></tr><tr><td> Fötales Kälberserum </td><td> Biosera </td><td> S9100 </td><td> Andere Quellen von fötalem Kälberserum in der Herstellung von TCM verwendet werden </td></tr><tr><td> EDTA </td><td> Fisher Scientific </td><td> BPE120-1 </td><td></td></tr></tbody></table>

detection of invasive pulmonary aspergillosis in haematological malignancy patients by using lateral-flow technology

Invasive pulmonale Aspergillose (IPA) ist eine führende Ursache für Morbidität und Mortalität bei Patienten mit hämatologischen Malignomen und hämatopoetischen Stammzelltransplantation Empfänger ein. Erkennung von IPA stellt eine beeindruckende diagnostische Herausforderung und, in Ermangelung eines &quot;Goldstandard&quot;, beruht auf einer Kombination von klinischen Daten und der Mikrobiologie und Histopathologie wo dies möglich ist. Die Diagnose der IPA sind an die Europäische Organisation für Forschung und Behandlung von Krebs und dem National Institute of Allergy and Infectious Diseases Mykologie Study Group (EORTC / MSG) Konsens zur Definition der &quot;bewährten&quot;, &quot;wahrscheinlich&quot; und &quot;möglich&quot;, invasive Pilzkrankheiten 2 entsprechen . Derzeit sind keine Nucleinsäure-basierten Tests wurden auf äußere IPA Detektion und so Polymerase-Kettenreaktion (PCR) validiert ist nicht im aktuellen EORTC / MSG diagnostischen Kriterien enthalten. Identifizierung von Aspergillus in histologischen Schnitten ist wegen der problematischen Simimäßigkeiten in Hyphen Morphologien mit anderen invasiven Pilzerreger 3 und bewährte Identifikation erfordert die Isolierung der Erreger in Reinkultur. Kultur-basierte Ansätze beruhen auf der Verfügbarkeit von Biopsie-Proben, aber diese sind nicht immer zugänglich in kranken Patienten, und nicht immer nachgeben lebensfähig Vermehrungseinheiten für Kultur, wenn erhalten. Ein wichtiges Merkmal in der Pathogenese von Aspergillus ist Angio-Invasion, eine Eigenschaft, die Chancen, den Pilz zu verfolgen immunologisch mittels Tests, die charakteristische Signaturen antigenen Moleküle nachzuweisen im Serum und in der bronchoalveolären Lavage (BAL) Flüssigkeiten bietet. Dies hat zu der Entwicklung der Platelia Enzymimmunoassay (GM-EIA), die Aspergillus Galactomannan und eine &quot;pan-Pilz&quot;-Assay (Fungitell Test), die den konservierten Zellwand-Komponente erkennt (1 → 3)-β-D-detektiert geführt Glucan, aber nicht in den Mucorales, die diese Komponente in ihren Zellwänden 1,4 fehlt. Istverklagt rund um die Genauigkeit dieser Tests 1,4-6 hat auf die jüngste Entwicklung der nächsten Generation monoklonaler Antikörper (MAK)-basierte Assays, die Surrogatmarker der Infektion erkennen 1,5 geführt. Thornton 5 Kürzlich beschrieb die Erzeugung eines Aspergillus-spezifischen mAb (JF5) unter Verwendung von Hybridom-Technologie und deren Verwendung, um eine Immun-chromatographisches Lateral-Flow-Gerät (LFD) für den Point-of-Care (POC) Diagnose von IPA zu entwickeln. Ein großer Vorteil des LFD ist seine Fähigkeit, die Aktivität zu erkennen, da MAb JF5 bindet an eine extrazelluläre Glykoprotein-Antigen, das während der aktiven Wachstum des Pilzes nur 5 sezerniert wird. Dies ist eine wichtige Überlegung bei der Verwendung von Flüssigkeiten, wie zB Lungen-BAL zur Diagnose IPA seit Aspergillus-Sporen ein gemeinsamer Bestandteil der Atemluft sind. Der Nutzen der Vorrichtung bei der Diagnose von IPA wurde gezeigt, mit einem Tiermodell der Infektion, wobei die LFD verbesserte Empfindlichkeit und Spezifikation angezeigtificity dem Platelia GM und Fungitell (1 → 3)-β-D-Glucan-Assays 7 verglichen. Hier stellen wir ein einfaches Verfahren, um LFD Aspergillus-Antigen in humanem Serum und BAL-Flüssigkeiten zu erkennen. Seine Geschwindigkeit und Genauigkeit bietet eine neuartige Ergänzung Point-of-Care-Test zur Diagnose von IPA in malignen hämatologischen Patienten.

Watch this Scientific Journal Video about Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology at JoVE.com

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Eine schnelle und genaue Point-of-Care-Test für invasiver pulmonaler Aspergillose wird vorgestellt. Es nutzt die Vorteile der Lateral-Flow-Technologie unter Verwendung eines spezifischen monoklonalen Antikörper, der an ein bindet<em&gt; Aspergillus</em&gt; Antigen bei Infektionen der Lunge ausgeschieden. Der Assay ist kompatibel mit Serum und brochoalveolar Lavage und stellt einen neuartigen Zusatz-Test zur Diagnose von Krankheitsbildern.

Nachweis des invasiven Pulmonale Aspergillose bei malignen hämatologischen Patienten mit Lateral-Flow-Technologie

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

detection-invasive-pulmonary-aspergillosis-haematological-malignancy

Research

JoVE Journal

Immunology and Infection

27.9K Aufrufe.  University of Exeter. Eine schnelle und genaue Point-of-Care-Test für invasiver pulmonaler Aspergillose wird vorgestellt. Es nutzt die Vorteile der Lateral-Flow-Technologie unter Verwendung eines spezifischen monoklonalen Antikörper, der an ein bindet Aspergillus Antigen bei Infektionen der Lunge ausgeschieden. Der Assay ist kompatibel mit Serum und brochoalveolar Lavage und stellt einen neuartigen Zusatz-Test zur Diagnose von Krankheitsbildern.

Serie: Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Nicht-invasive Bildgebung von Leukozyten Homing und Migration In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Forschung

Immunologie und Infektion

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Kombination von Klebstoff-tape-basierten Sampling und Fluoreszenz In situ Hybridisierung zum schnellen Nachweis von Salmonellen On Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Nachweis von Bakterien mit fluorogenen DNAzyme

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Nachweis von microRNAs in Mikroglia durch Real-time PCR in Normal-und ZNS Während Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

In Vivo Imaging Systems (IVIS) Nachweis eines Neuro-Invasive encephalitische Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

Rechtsventrikulären systolischen Druck Messungen in Kombination mit Harvest of Lung and Immune Gewebeproben in Mäuse

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

Verfahren zur Erzeugung von Lungen Neutrophilie Mit aerosolisiertes Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

Der Nachweis von Human-Leukozyten-Antigen Biomarkern bei Brustkrebs Verwendung Markierungsfreie Biosensorik

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Hochdurchsatz-Nachweis von Respiratory Pathogens in Tier Proben durch nanoskalige PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Generation of Murine Monoclonal Antibodies by Hybridomtechnologie

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...