Name: automated acoustic dispensing for the serial dilution of peptide agonists in potency determination assays
Uploaded: 2016-11-10T12:30:00.000+00:00
Duration: 8 min 6 s
Description: Watch this Scientific Journal Video about Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays at JoVE.com

1. Peptidserienverdünnung <ol><li> Bereiten Assaypuffer: Hanks gepufferter Salzlösung (HBSS), supplementiert mit 25 mM HEPES, 0,1% BSA und 0,5 mM 3-Isobutyl-1-methylxanthin (IBMX), pH 7,4. </li><li> Verwenden Sie einen Bulk-Reagenzspender systematisch 5 ul Testpuffer hinzufügen zu jeder Vertiefung von fünf 384-Well-Low-Volume-Testplatten. <ol><li> Verwenden Sie den internen Software ein Abgabeprogramm für 5 ul Volumen zusätzlich zu jeder Vertiefung einer 384-Well-Platte zu erstellen, wie Anweisungen des Herstellers. </li><li> Tauchen Abgabe Kassettenschläuche in Testpuffer und Prime Flüssigkeit. </li><li> Legen Sie 384-well geringem Volumen Assayplatte auf Plattenträger. </li><li> Drücke Start. </li></ol></li><li> Verdünnen Sie alle Peptidproben, unabhängig von Speicher Fahrzeug, in Testpuffer ein 100x Peptid Lager herzustellen. HINWEIS: Peptides Screening erfordern , können in phosphatgepufferter Salzlösung (PBS) oder Dimethylsulfoxid (DMSO) bereitgestellt werden , wie als angemessen erachtet wird (beispielsweise DMSO wkrank haben eine schädliche Wirkung auf Peptide mit sekundären Modifikationen wie PEGylierung). </li><li> Dispense 25 ul 100x Peptid Bestände in Spalten 1-5 von einem akustisch qualifizierten 384-Mikrotiterplatte aus Polypropylen. Diese Platte ist &#39;bezeichnet Source-Platte A&#39;. Stellen Sie sicher, dass die Quellplatten, aber nicht Zielplatten sind mit flachem Boden und entsprechen den spezifischen akustischen Toleranzen, wie vom Hersteller von akustischen Instrumenten definiert. </li><li> Dispense 25 ul 100x Referenzkontrolle in die Vertiefungen A23 und A24 von Quellenplatte A. </li><li> Dispense 10 &amp; mgr; l Testpuffer in Spalten 11 bis 15 und 30 &amp; mgr; l Testpuffer in Spalten 21-22 von Quellenplatte A. </li><li> Dispense 10 &amp; mgr; l Testpuffer in Spalten 6-10 und Spalten 16-20 eines zweiten akustisch qualifiziert 384-Mikrotiterplatte aus Polypropylen. Diese Platte wird &quot;Quellenplatte B &#39;bezeichnet. </li><li> Zentrifuge Quellenplatten A und B bei 300 × g für 1 min. Fügen Sie eine entsprechende Ausgleichsplatte. </li><li> Verwenden Sie akustische fluid Spender in ein automatisiertes Robotersystem integriert drei aufeinanderfolgenden 1 vorzubereiten: aus der Kolonne 100 Zwischenverdünnungen (in Testpuffer) der 100x-Peptid-Aktien 1 in Quelle A. HINWEIS: Die Programmierung erfordert Platte neu formatiert und Dosis - Wirkungs - Software (wie vom Hersteller des akustischen Flüssigkeitsspender zur Verfügung gestellt) drei akustische Übertragungen zwischen Quelle A und Quelle B zu ermöglichen (Abbildung 1 für Plattenlayouts sehen). Schritt 1.9 Details insbesondere die Verwendung von Fluidspender in diesem Labor unter automatisierten Robotersteuerung verwendet wird (siehe Materialien Table): <ol><li> Ladequellenplatten und Testplatten in Plattenhotelteil der Robotik. </li><li> Offene automatisierten Roboter-Software und Lastplatte neu formatiert und Dosis-Wirkungs-Programmerweiterung Protokolle. Klicken Sie auf &quot;Ausführen&quot;. HINWEIS: Automation weist einen akustischen Flüssigkeitsspender 250 nl von Spalten A 1-5 von Quellenplatte in Spalten zu übertragen 6-10 von Quellenplatte B und eine zweite Acoustic Flüssigkeitsspender der Lage, größere Flüssigkeitsvolumina Umgang mit 15 &amp; mgr; l Testpuffer zu verfüllen zu mischen. Automation überträgt dann Source-Platte B zu integrierten Zentrifuge und Zentrifugen bei 300 g für 1 min (eine entsprechende Ausgleichsplatte ist für alle Zentrifugationsschritte enthalten). Source Platte B ist mit dem akustischen Fluidspender für die Übertragung von 250 nl zurück von Spalten 6-10 der Quellenplatte B in Spalten 11-15 der Quellenplatte A und Verfüllung mit 15 ul Assaypuffer zu mischen. Automation überträgt Quellenplatte A integrierte Zentrifuge und Zentrifugen bei 300 g für 1 min. Quelle Platte A wird dann zu dem akustischen Flüssigkeitsspender für den Transfer von 250 nl von Spalten 11-15 der Quellplatte A in Spalten 16-20 der Quellplatte B und Verfüllung mit 15 &amp; mgr; l Testpuffer zu mischen. Automation überträgt dann Source-Platte B zu integrierten Zentrifuge und Zentrifugen bei 300 g für 1 min. HINWEIS: Schließlich wird die Dosis-Wirkungs-Protokoll leitet akustische Ausgabe zu übertragendas erforderliche Volumen von jedem der vier seriell Source-Platte Brunnen verdünnt (sowohl in der Source-Platte A und Source-Platte B) 1.2 eine vollständige 11-Punkt-Kurve in zweifacher Ausfertigung in Assayplatten (gefüllt mit 5 &amp; mgr; l Testpuffer in Schritt zu konstruieren oben ). Automation Transfers Assayplatte integrierte Zentrifuge und Zentrifugen bei 300 g für 1 min. </li></ol></li></ol> 2. Zellpräparation <ol><li> Auftau kryokonservierten Chinese hamster ovary (CHO) Zellen, die das Ziel Maus GLP-1-Rezeptor rasch in einem 37 ºC Wasserbad und Resuspendieren in 20 ml Assaypuffer exprimieren. </li><li> Zentrifuge Zellsuspension für 5 min bei 200 xg bei Raumtemperatur (RT). Fügen Sie ein angemessenes Gleichgewicht. </li><li> Überstand verwerfen und resuspendieren Zellpellet in 10 ml Assaypuffer. </li><li> Verdünnen Zelle Lager 1: 1 in Trypanblau und bestimmen Lebendzelldichte eine automatische Zellzähler verwendet wird. Die Zellen in Assaypuffer bei 1,6 x 10 6 Zellen pro ml (äquivalent zu 8000 Zellen pro well Assayplatten). </li><li> Verwenden einer Schütt Reagenzspender 5 &amp; mgr; l der Zellsuspension hinzugefügt zu jeder Vertiefung der Testplatten (mit seriell verdünnten Peptide in 5 &amp; mgr; l Assay-Puffer) und Inkubation für 30 min bei RT. </li></ol> 3. HTRF cAMP-Detection-Assay <ol><li> Bringen HTRF cAMP-Assay-Kit für 30 min auf RT vor der Verwendung. </li><li> Bereiten Sie jede HTRF-Reagenz (Kryptat und d2) getrennt bei einer Verdünnung von 1:20 in Lysispuffer (eigene Formulierung, durch Hersteller von cAMP-Nachweistest zur Verfügung gestellt). HINWEIS: ACHTUNG: Lysepuffer enthält Kaliumfluorid (KF) , die toxisch ist und einen teratogen 13. Zur Entsorgung Assay-Platten, die KF sollte versiegelt und verbrannt werden, und jede flüssige Abfälle müssen mit Wasser vor der Entsorgung in den Ausguss auf &lt;1 mmol / l verdünnt werden. </li><li> Verwenden Sie einen Bulk-Reagenzspender 5 ul Kryptat- Reagenz in alle Vertiefungen der Testplatten hinzuzufügen. </li><li> Verwenden Sie einen Bulk-Reagenzspender 5 ul d2 Reagenz colum hinzufügenns 1-22 der Testplatten. </li><li> Unmittelbar Pipette manuell 5 &amp; mgr; l Lyse-Puffer in Vertiefungen A23-D24 der Testplatten nicht-spezifische Bindung (NSB) Kontrollvertiefungen zu geben, und 5 ul d2 Reagenz in die Vertiefungen E23-P24. </li><li> Zentrifugieren Sie die Testplatten für 1 min bei 200 × g bei RT die Brunnen zu mischen. </li><li> Decken Sie die Testplatten Verdunstung und Photobleaching zu minimieren und Inkubation bei RT für 1 Stunde. </li><li> Messen Sie Fluoreszenz-Resonanz-Energie-Transfer (FRET) Signal mit einer Anregung bei 320 nm und Emission bei 620 nm und 665 nm einen Plattenleser. </li></ol> 4. Datenanalyse <ol><li> Verwenden Sie NSB bedeuten Hintergrund aus allen Wells abzuziehen. </li><li> Berechnen% Delta F aus dem 665 nm / 620 nm-Verhältnis wie folgt: Ratio = (A 665 nm / A 620nm) x 10 4 Delta F = ((Sample Ratio - Verhältnis NSB) / Ratio - NSB)) x 100 wo Verhältnis NSB = Vertiefungen ohne d2 Reagenz. </li><li> Berechnen% betätigendesIonen zwischen unstimulierten Zellen und Zellen mit maximaler GLP-1-Liganden stimuliert, wie folgt: % Aktivierung = (% Delta fSAMPLE -% DeltaFunstimulated Zellen) / (% DeltaFGLP-1 stimulierten Zellen -% DeltaF unstimulierten Zellen) x 100 </li><li> Analysieren Sie Konzentrations-Wirkungs - Kurven über 4-Parameter logistische Analyse und grafische Darstellung in% Aktivierung , wie durch Bezugssteuerung 1 definiert. </li></ol>

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The authors have nothing to disclose.

Dieses Protokoll beschreibt die erfolgreiche Anwendung von automatisierten akustischen Ausgabe zu seriell Peptidproben über einen Konzentrationsbereich von 3 x 10 6 , die weniger als 1 ul Probe verdünnen. Der große Vorteil dieser Methode ist, über reduzierte Exposition von Proben zu experimentellen Behälter und Kunststoffgeschirr (wie Pipettenspitzen) Datenqualität durch Minimierung Peptid Adsorption an Kunststoffprodukte zu erhöhen, die für Reagenztransfer und Mischen normalerweise erforderlich sind....

Die GLP-1R ist ein etabliertes Medikament Ziel bei der Behandlung von Typ - 2 - Diabetes 2. Das native Peptid - Agonisten für diesen Rezeptor, GLP-1, hat eine in vivo Halbwertszeit von 2-3 min 3. Die Bindung von GLP-1 an seinen G-Protein-gekoppelten Rezeptorziel Ergebnisse in der stromabwärtigen Herstellung des second messenger cAMP durch native G-Protein-Kopplung an die Aktivierung von Adenylatcyclase. Die Messung des akkumulierten cAMP stellt eine robuste Test-Rezeptoraktivierung zu überwachen und für die aktive GLP-1-Analoga mit bevorzugten physikalisch-chemischen Eigenschaften zu screenen. Ein solcher Assay erfordert die serielle Verdünnung der Testproben Konzentrations-Reaktions-Kurven zu konstruieren, und dies ist besonders kompliziert, wenn Peptidproben reichte. Mögliche Fehler von der Spitze-basierten seriellen Verdünnung Vorbereitung wurden zuvor 1,4,5 beschrieben. Peptide werden auf Kunststoffgeschirr, was zu unzuverlässigen Potenz Schätzungen adsorbieren. Peptidverlust kann durch t minimiert werdener die Aufnahme von Rinderserumalbumin (BSA) in Puffer und die Verwendung von silikonisierten Kunststoffgeschirr, noch Proteinbindungs ​​bleibt unvorhersehbar. Insbesondere wurde die Änderung der Bindung von GLP-1 an Versuchsbehältern wurde 6 beschrieben. Es gibt eine weitere Komplikation bei , dass die Stabilisierungsmittel in Laborkunststoffprodukte verwendet wird, kann von den Spitzen und Mikrotiterplatten in wßriges Assaypuffer auslaugen und stören Proteinfunktion 7, 8. Daher Methoden Exposition gegenKunstStoffGeschirr zu reduzieren , sind notwendig , um die Genauigkeit der Messungen zu erhöhen. Akustische Flüssigkeitsspender konzentriert eines hochfrequenten akustischen Signals auf der Oberfläche einer Flüssigkeitsprobe, wodurch der Ausstoß von Tröpfchen präzise Nanoliter- in eine benachbarte Testplatte 9. Die Verwendung von akustischen Ausstoß ist in der pharmazeutischen Industrie für die Herstellung und das Screening von großen synthetischen Substanzbibliotheken Standard, und die Technologie für kleine molecul gut validiert wurdees 10. Unseres Wissens sind wir die erste Gruppe akustische Abgabe zur Herstellung von rekombinanten und synthetischen Peptide zu beschreiben , und wir haben bereits berichtet , die eine verbesserte Genauigkeit im Vergleich zu herkömmlichen Tipp-basierten Methoden 1. Dieser Artikel beschreibt die Integration der Herstellung von Peptid serielle und direkte Verdünnungen von berührungslosen akustischen Übertragung auf eine vollautomatische Plattenrobotersystem Handhabung. Eine Anzahl von Verfahren umfasst Akustisches von Proben wurden zuvor 11 beschrieben. Wir verwenden ein zweistufiges Verfahren zur Zwischenstammkonzentrationen herzustellen und zu seriell Peptidanaloga für die Erzeugung der vollen Dosis-Wirkungskurve verdünnen. Die hergestellten Peptide werden inkubiert mit den Zellen des Ziel Maus GLP-1R exprimieren, und wir verwenden eine im Handel erhältliche homogene zeitaufgelöste Fluoreszenz (HTRF) -Assay cAMP-Akkumulation in diesen Zellen als eine Auslesung von Peptid-Agonist aktiv zu messen,keit. Der Assay ist robust und zugänglich für ein Hochdurchsatz - 384-Well - Format und routinemäßig sowohl Assay - Entwicklung angewendet und Wirkstoff - Screening - Projekte 12.

<table><tbody><tr><td>Hanks&amp;rsquo; Balanced Salt solution</td><td>Sigma-Aldrich</td><td>H8264</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I7018</td><td>Prepared as a 0.5 M stock in DMSO</td></tr><tr><td>GLP-1 (7-36) amide</td><td>Bachem</td><td>H-6795</td><td>Prepared as a 1 mg/ml stock in PBS, referred to as &#039;100x reference control&#039;</td></tr><tr><td>Test peptides</td><td>Produced in-house at MedImmune</td><td></td><td>Supplied at various concentrations in DMSO or PBS as appropriate</td></tr><tr><td>100x peptide stock</td><td>Produced in-house at MedImmune</td><td></td><td>Test peptide diluted into assay buffer to 100x final required concentration</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250-061</td><td></td></tr><tr><td>Cedex XS Cell Analyzer</td><td>Innovatis</td><td></td><td></td></tr><tr><td>Corning 384 well plates, low volume</td><td>Sigma-Aldrich</td><td>4514</td><td></td></tr><tr><td>Echo Qualified 384-Well Polypropylene Microplate</td><td>Labcyte Inc.</td><td>P-05525</td><td></td></tr><tr><td>Echo Qualified Reservoir</td><td>Labcyte Inc.</td><td>ER-0055</td><td></td></tr><tr><td>Echo 550 Liquid Handler</td><td>Labcyte Inc.</td><td></td><td>Droplet transfer volumes in increments of 2.5 nl</td></tr><tr><td>Echo 525 Liquid Handler</td><td>Labcyte Inc.</td><td></td><td>Droplet transfer volumes in increments of 25 nl</td></tr><tr><td>ACell Benchtop Automation&amp;nbsp;</td><td>HighRes Biosolutions</td><td>MC522</td><td></td></tr><tr><td>Cellario Lab Automation Scheduling software for Life Science Robotics</td><td>HighRes Biosolutions</td><td></td><td></td></tr><tr><td>MultidropCombi Reagent Dispenser</td><td>ThermFisher Scientific</td><td>5840300</td><td>Referred to as &#039;bulk reagent dispenser&#039;</td></tr><tr><td>HTRF cAMP Dynamic 2 kit</td><td>Cisbio Bioassays</td><td>62AM4PEJ</td><td></td></tr><tr><td>EnVision Multilabel Reader</td><td>PerkinElmer</td><td></td><td></td></tr></tbody></table>

automated acoustic dispensing for the serial dilution of peptide agonists in potency determination assays

Wie bei Entdeckung kleines Molekül als Wirkstoff, erfordert das Screening für Peptid-Agonisten die serielle Verdünnung von Peptiden Konzentrations-Wirkungs-Kurven zu erzeugen. Screening Peptide bietet eine zusätzliche Ebene der Komplexität als herkömmliche Spitzenbasis Probenhandhabungsverfahren Peptide zu einer großen Oberfläche von Plastikmaterialien belichten, eine erhöhte Chance für Peptidverlust durch Adsorption bereitstellt. Verhindern übermäßigen Exposition gegenüber Kunststoffgeschirr reduziert Peptidverlust durch Festhalten an Kunststoffen und minimiert somit Ungenauigkeiten in Potenz Vorhersage, und wir haben zuvor beschrieben die Vorteile von akustischen berührungslosen Abgabe für in vitro High-Throughput - Screening von Peptidagonisten 1. Hier diskutieren wir eine vollständig integrierte Automatisierungslösung zur berührungslosen akustischen Herstellung von Peptid-Reihenverdünnungen in Mikrotiterplatten unter Verwendung der beispielsweise zum Screenen von Peptidagonisten an der Maus Glucagon-like Peptid-1-Rezeptor (GLP-1R). Unsere Methoden ermöglichen hohe-throughput zellbasierten Assays zum Screenen auf Agonisten und sind leicht skalierbar erhöhten Probendurchsatz zu unterstützen, oder für eine erhöhte Anzahl von Kopien Testplatte (beispielsweise für eine Gruppe von mehreren Zielzelllinien) zu ermöglichen.

<html> Wir verwenden routinemäßig einen zweistufigen Verfahren Peptide über akustische Übertragung zu verdünnen. Für den ersten Schritt wird ein akustisches Spender mit Automatisierungs fluchtet verwendet vier Stammpeptidzwischenverdünnungen über zwei Quellenplatten (1a, b) zu erzeugen. Für den zweiten Schritt, verwenden wir einen akustischen Spender zur weiteren Stammverdünnungen von der Quelle Platten A verdünnen und B eine 11-Punkt - Konzent...

Watch this Scientific Journal Video about Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays at JoVE.com

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

Automatische Acoustic Verzicht für die serielle Verdünnung von Peptid-Agonisten im Potency Bestimmung Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

automated-acoustic-dispensing-for-serial-dilution-peptide-agonists

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

7.6K Aufrufe.  MedImmune. Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described. 

Serie: Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

Peptid und Protein Quantifizierung mit Hilfe automatisierter Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Forschung

Chemie

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Drug-induzierte Sensibilisierung der Adenylatzyklase: Assay Straffung und Miniaturisierung für Small Molecule-und siRNA-Screening-Anwendungen

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Biotechnik

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impeddance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

Biochemie

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Skalierbare High Throughput Auswahl aus Phagen-Antikörperbibliotheken angezeigt Synthetische

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Immunologie und Infektion

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

Ein "Dual-Addition"-Calciumfluoreszenz-Assay für das Hochdurchsatz-Screening von rekombinanten G-Protein-gekoppelten Rezeptoren

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of applications. The cell surface proteome ("surfaceome") in human disease is of significant interest, as plasma membrane proteins are the primary target of most clinically approved therapeutics, as well as a key feature by which to diagnostically distinguish diseased cells from healthy tissues. However, focused characterization of membrane and surface proteins of the cell has remained challenging, primarily due to the complexity of cellular lysates, which mask proteins of interest by other high-abundance proteins. To overcome this technical barrier and accurately define the cell surface proteome of various cell types using mass spectrometry proteomics, it is necessary to enrich the cell lysate for cell surface proteins prior to analysis on the mass spectrometer. This paper presents a detailed workflow for labeling cell surface proteins from cancer cells, enriching these proteins out of the cell lysate, and subsequent sample preparation for mass spectrometry analysis.

Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software.

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Synthese von Monocyte-targeting Peptid Amphiphile Micellen für die Bildgebung der Atherosklerose

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Quantifizierung von Proteinen über ein Peptid Immunoaffinity Enrichment gekoppelt mit der Massenspektrometrie

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Biologie

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Ein hoher Durchsatz MHC II-Bindungsassay für die quantitative Analyse von Peptid-Epitope

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Einfache Massen Auslesen des digitalen Nukleinsäure Quantifizierung Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...