The authors thank Miss Ching-Chun Lin and Miss Diana Lin of the Core Facility of the Institute of Cellular and Organismic Biology (ICOB) of Academia Sinica for offering their expertise on peptide synthesis and DNA sequencing, respectively. This study was supported by Academia Sinica.

1. Herstellung des monoklonalen Antikörpers <ol><li> Kultur die RG-M56 monoklonalen Maus - Hybridomzellen 2 in Serum-freiem Medium in 175T - Kolben bei 37 ° C mit 5% CO 2 zu ergänzen. Die überstehende Flüssigkeit, wenn die Farbe des Mediums nach fünf Tagen Inkubation gelb wird. HINWEIS: Hybridoma-Zellen wurden in serumfreiem Medium Antikörper Kontamination aus fötalem Rinderserum zu vermeiden. </li><li> Zentrifugieren Sie den Überstand bei 4.500 × g für 30 min bei 4 ° C und entsorgen die Zelltrümmer Pellet. </li><li> In 2 ml Protein G-Agarose (geliefert als eine 50% ige Aufschlämmung) auf eine 5-ml-Säule und ins Gleichgewicht mit 10 Harzvolumina (10 ml) eiskaltem PBS. </li><li> Legen Sie 200 ml des Antikörpers Überstand (Schritt 1.2) auf die Säule und entsorgen Sie die Pass-Through. </li><li> 10 ml eiskaltem PBS auf die Säule zu waschen. Zweimal wiederholen. </li><li> 10 ml 50 mM Glycin, pH 2,7 auf die Säule, die das Protein G-assoziierten Antikörper zu eluieren. ColLect 900 ul-Fraktionen in einem Mikrozentrifugenröhrchen, das 100 ul 10fach Neutralisationspuffer (1 M Tris, 1,5 M NaCl und 1 mM EDTA, pH 8,0). </li><li> Lagern Sie das gereinigte Antikörper in 50% Glycerin mit 0,03% NaN 3 bei -20 ºC. </li></ol> 2. Konstruktion und Expression von Abwechselnd Verkürzte rekombinanten Proteine <ol><li> Bereiten Sie eine PCR - Reaktionsgemisch: 5 ul 10x Pfu - Puffer, 0,2 mM von jedem dNTP, 0,2 &amp; mgr; M Vorwärtsprimer 3, 0,2 uM Reverse - Primer 3, 2 mM MgSO 4, 1 ng pET20b-1A59 3 Plasmid - DNA und 2,5 U ( Einheit) von Pfu DNA - Polymerase; ddH 2 O auf ein Endvolumen von 50 &amp; mgr; l hinzuzufügen. <ol><li> Führen Proben in einem automatischen Thermocycler mit folgenden Parametern: Zyklus 1 (94 ° C für 5 min); 2-36 Zyklen (94 ° C für 30 s, 63 ºC für 30 s und 72 ° C für 60 s); und Zyklus 37 (72 ºC für 7 min). </li></ol></li><li> Extrahieren der Polymerasekettenreaktion (PCR) Produkte durch eine PCR - Reinigungskits 5 unter Verwendung der folgenden Restriktionsenzym - Verdau zu ermöglichen. </li><li> Digest die PCR-amplifizierten DNA - Fragmente mit Nde I und Xho I - Restriktionsenzymen und Ligation jedes dieser DNA - Fragmente in Nde I und Xho I gespalten enzym pET-20b (+) Vektor. Verwandeln Sie die Konstrukte in Escherichia coli DH-5α kompetenten Zellen 6. <ol><li> Bereiten Sie die Aufschlussmischung in Verdauungspuffer (20 mM Tris-Acetat, 10 mM Mg (CH 3 COO) 2, 50 mM KCH 3 COO und 1 mM DTT, pH 7,9) mit 1 ug PCR-amplifizierte DNA oder pET-20b (+) Vektor - DNA und 2 U von Nde I und Xho I - Restriktionsenzymen in einem Endvolumen von 20 &amp; mgr; l. </li><li> Mischen Sie die Verdauung Mischung sanft und schnell drehen nach unten. bei 37 ºC inkubieren für 2 h in einem trockenen Bad die komplette Schneiden der Einschränkung, um sicherzustellen, sitzenes. </li><li> Extrahieren Sie die Restriktionsenzym-verdauten DNA - Fragmente unter Verwendung eines PCR - Reinigungs - Kit 5 , um die folgenden Plasmidkonstruktion erleichtern. </li><li> Bereiten Sie die Ligierungsmischung in Ligationspuffer (66 mM Tris, 5 mM MgCl 2, 1 mM ATP und 5 mM DTT, pH 7,5) mit 100 ng predigested, PCR-amplifizierte DNA, 10 ng vorverdaut pET-20b (+) Vektor-DNA und 5 U des T4 DNA-Ligase in einem Endvolumen von 10 &amp; mgr; l. </li><li> Mischen Sie das Ligierungsgemisch schonend und schnell Spin-Down. bei 16 ºC inkubieren für 18 Stunden in einem Wasserbad. </li><li> Setzen Sie 10 ul der Ligationsproben in 100 &amp; mgr; l der DH-5α kompetenten Zellen und vorsichtig mischen, bevor das Mikrozentrifugenröhrchen auf Eis für 30 min platzieren. Setzen Sie den Mikrozentrifugenröhrchen in einem trockenen Bad bei 42 ° C für 90 s Hitzeschock 7 zu induzieren. Unmittelbar übertragen das Rohr auf Eis für 2 min. </li><li> Hinzufügen 900 uL Luria-Bertani (LB) Medium (1% Bacto Trypton, 0,5% Bacto Hefe extract, und 0,5% NaCl, pH 7,0) in das Röhrchen. bei 37 ºC inkubieren mit 150 Umdrehungen pro Minute für 45 Minuten schütteln. Pellet die Zellen durch Zentrifugation bei 4000 x g für 10 Minuten und den Überstand verwerfen. </li><li> Resuspendieren des Pellets mit 50 &amp; mgr; l LB-Brühe und verteilt jede Transformation auf vorgewärmte LB-Platten, enthaltend 100 ug / ml Ampicillin. Die Inkubation bei 37 ° C für 16 Stunden. </li><li> Pick-up eine einzelne Kolonie mit einer 200 ul Spitze und legen Sie sie in 3 ml LB-Brühe, die 100 ug / ml Ampicillin in einem lose verschlossen 15 ml-Röhrchen. bei 37 ºC inkubieren mit 150 Umdrehungen pro Minute für 12 h unter Schütteln. </li><li> Extrahieren Sie die Plasmid - DNA 8 aus jeder Kultur und sequenzieren es T7 - Promotor und T7 - Terminator - Primer unter Verwendung der Sequenz 9 zu bestätigen. </li></ol></li><li> Nach Sequenzbestätigung, 10 ng DNA aus diesen mit verschiedenen Längen von YGNNV Hüllprotein - Gen in den BL-21 (DE3) -Stamm von E. coli durch uns pET-20b (+) Plasmide transformierening die Hitzeschock - Methode 7. Führen Sie die Schritte 2.3.6-2.3.8 die Transformation durchzuführen. </li><li> Übertragen einer einzelnen Kolonie aus jeder transformierten E. coli BL-21 - Zellen in 3 ml LB - Brühe , die 100 ug / ml Ampicillin in einem lose capped 15 ml - Röhrchen. Inkubieren Sie die Kultur bei 37 ° C mit 150 Umdrehungen pro Minute schütteln. </li><li> Man kühlt die Kultur auf 25 ºC , wenn die OD 600 der Kultur etwa 0,6 ist und fügen IPTG bis zu einer Endkonzentration von 0,4 mM , die Expression des rekombinanten Proteins zu induzieren. Inkubieren Sie die Kultur für eine zusätzliche 4 h bei 25 ° C mit 200 Umdrehungen pro Minute schütteln. </li><li> 1 ml der Kultur in ein Mikrozentrifugenröhrchen. Pellet die Zellen durch Zentrifugation bei 12.000 × g für 1 min und den Überstand verwerfen. </li><li> Zellpellet in 100 ul Denaturierungspuffer (8 M Harnstoff, 20 mM Natriumphosphat und 0,5 M NaCl, pH 7,4) durch Auf- und Abpipettieren mit einer Mikropipette. Mischen durch kräftiges Vortexen. HINWEIS: Die Probe solutions sollte nun semi-transparent, ein wenig klebrig, und bereit für die Dot-Blot-Hybridisierungstest ist. </li></ol> 3. Entwurf und Synthese von überlappenden Peptiden <ol><li> Design und Synthese von 3 serielle 20-mer Peptide , die jeder mit seinem Nachfolger überlappen um 10 AS - Reste aus dem 195-338 aa Bereich des YGNNV Hüllprotein des Epitopbereichs von RG-M56 mAb durch Dot - Blot zu verengen. </li><li> Design und Synthese von 3 drei 8-mer - Peptide (195 VNVSVLCR 202, 197 VSVLCRWS 204 und 199 VLCRWSVR 206) mit einer Überlappung von 6 AS - Reste auf die nächste synthetisches Peptid , das das Epitop Region von 195-206 aa durch Dot - Blot zu verengen. </li><li> Design und Synthese von 3 7-mer (196 NVSVLCR 202 und 195 VNVSVLC 201), 6-mer (195 VNVSVL 200, 196 NVSVLC 201 und 197 VSVLCR 202) und 5-mer - Peptide (195 VNVSV 199, 196 NVSVL 200, 197 VSVLC 201 und 198 SVLCR 202) mit einer Überlappung von 6, 5 und 4 aa - Reste jeweils an ihren benachbarten Peptide die zur Minimierung Epitopbereichs durch dot-Blot. </li></ol> 4. Dot-Blot-Hybridisierung <ol><li> Auflösen jedes synthetisierte Peptid in Dimethylsulfoxid (DMSO) auf eine Endkonzentration von 10 mg / mL. HINWEIS: Um die vielfältigen Löslichkeit von synthetischen Peptiden zu überwinden, alle synthetischen Peptide sollten in DMSO gelöst werden. DMSO ist ein gutes Lösungsmittel vollständig hydrophob oder hydrophile Peptide lösen. </li><li> Genießen Sie die Polyvinylidenfluorid (PVDF) Membran mit Methanol für 2 min. HINWEIS: PVDF-Membran ist bis zu 100% beständig DMSO; andere können nicht so sein. </li><li> Äquilibrieren die PVDF-Membran mit modifizierten Towbin-Puffer (25 mM Tris, 192 mM Glycin und 0,1% SDS, pH 8,3) für2 Minuten. HINWEIS: 10-20% (v / v) Methanol kann modifiziert Towbin Puffer hinzugefügt werden die Übertragungsergebnisse zu verbessern. </li><li> Spülen Sie ein Stück Papier Chromatographie mit dem modifizierten Towbin-Puffer. Legen Sie die PVDF-Membran auf die Chromatographie-Papier. Warten, bis der modifizierte Towbin Puffer aus der PVDF Membranoberfläche verschwunden ist, bevor mit dem nächsten Schritt fort. </li><li> Fügen Sie 2 ul jeder Peptidprobe an die Membran mit einer 10 &amp; mgr; l Spitze. Der Luft trocknen die PVDF-Membran auf der Chromatographie-Papier für 10 min. Fügen Sie jede Peptidprobe langsam und allmählich auf die Membran zu viel Diffusion zu vermeiden. </li><li> Blockieren die Membran in TBST-Puffer (0,05% (v / v) Tween-20, 20 mM Tris und 150 mM NaCl, pH 7,4) mit 5% Magermilch 30 min bei Raumtemperatur unter leichtem Schütteln inkubiert. </li><li> Hinzufügen RG-M56 mAb bei einer Endverdünnung von 1: 1000 in TBST-Puffer mit 5% fettfreier Milch in die Membran. Inkubieren Sie die Membran bei 37 ºC für 1 h unter leichtem Schütteln. </li><li> Entfernen Sie die Antikörper solution. Waschen Sie die Membran in TBST-Puffer für 5 Minuten unter leichtem Schütteln. Zweimal wiederholen. </li><li> Fügen der sekundäre Antikörper (Ziege-anti-Maus IgG, Fc, konjugiert mit alkalischer Phosphatase) in einer Endverdünnung von 1: 5000 in TBST-Puffer mit 5% fettfreier Milch in die Membran. Inkubieren Sie die Membran bei 37 ºC für 1 h unter leichtem Schütteln. </li><li> Entsorgen Sie die Antikörperlösung. Waschen Sie die Membran in TBST-Puffer für 5 Minuten unter leichtem Schütteln. Wiederholen Sie den Waschschritt zweimal. </li><li> Entwickeln der Membran mit BCIP / NBT-Substratlösung während 15 min im Dunkeln bei Raumtemperatur. Stoppen Sie die Entwicklung durch die Membran mit ddH 2 O gewaschen , wenn das Signal erscheint. </li><li> Der Luft trocknen die Membran und erfassen die Dot-Blot-Bild ein Bild System. </li><li> Messen der Intensität jedes Dot - Blot unter Verwendung von Bildanalysesoftware 3. </li></ol> 5. Alaninscanning und Substitution <ol><li> Entwerfen und zu synthetisieren 3 Alanin und methionine Substitutionspeptide. Ersetzen Sie jede aa Rückstand mit Alanin für die 8-mer Peptid 195 VNVSVLCR 202 und synthetisieren Peptide 195 ANVSVLCR 202, 195 VAVSVLCR 202, 195 VNASVLCR 202, 195 VNVAVLCR 202, 195 VNVSALCR 202, 195 VNVSVACR 202, 195 VNVSVLAR 202 und 195 VNVSVLCA 202 . Ersetzen aa Rest Leucin 200 zu Methionin das SJNNV Genotyp - Epitop - Peptid 195 VNVSVMCR 202 zu schaffen. </li><li> Folgen Sie Abschnitt 4 zu Alanin-Scanning und Substitutionsmutagenese durchführen Dotblotting. </li></ol>

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The authors have no conflicts of interest related to this report.

Dieses Protokoll bietet eine schnelle und einfache Technik, um ein mAb anerkannte lineares Epitop zu identifizieren. Unter Berücksichtigung der Kosten für die Peptidsynthese und die Produktionseffizienz der Synthese von Peptiden wurde die antigene Region des Virus-Hüllproteins reduziert durch serielles trunkierten rekombinanten Proteinen vor Peptid-Scanning-Analyse exprimieren. Als solche wurde die zuverlässig und effizient E. coli pET Expressionssystem diese seriell trunkierten rekombinanten Proteine als r...

Im Immunsystem, die Rekombination von V-, D- und J - Segmente können für Antikörper für die Bindung an verschiedene Antigene enorme Variationen von komplementaritätsbestimmenden Regionen (CDRs) zu erzeugen , die Host von pathogenen Infektion zu schützen. Die neutralisierende Abwehr von Antikörpern gegen Antigene hängt von der räumlichen Komplementarität zwischen den CDRs der Antikörper und die Epitope der Antigene. Daher wird ein Verständnis dieser molekularen Wechselwirkung prophylaktischen Impfstoff-Design und therapeutischen Peptid Arzneimittelentwicklung unterstützen. Jedoch kann diese Neutralisation Wechselwirkung sowohl aus einem einzigen Antigen und durch mehrere CDRs von Antikörpern durch multiple antigene Domänen beeinflußt werden, die somit das Epitop Bestimmungsprozess komplexer machen. Glücklicherweise ist die Entwicklung der Hybridom-Technologie, die einzelnen Antikörper-produzierenden Zellen mit Myelomzellen verschmilzt, ermöglicht eine ständig Dividieren Charge von Zellen secrete einem spezifischen Antikörper, bekannt als ein monoklonaler Antikörper (mAb) 1. Hybridomzellen produzieren diese reine, hochaffine mAb an einem einzigen Antigen-Domäne eines spezifischen Antigens zu binden. Mit der Beziehung des Antigen-Antikörper festgestellt, mehrere Ansätze, einschließlich Peptid-Scanning, verwendet werden, um das Epitop eines Antigens zu bestimmen seinen entsprechenden mAb verwendet. Die jüngsten Entwicklungen in der synthetischen Peptid-Technologie haben die Peptid-Scanning-Technik zugänglicher und bequemer gemacht auszuführen. Kurz gesagt, werden gemäß einer Zielantigensequenz einen Satz von überlappenden synthetischen Peptiden hergestellt und zu einem Feststoff-Trägermembran für mAb Hybridisierung verbunden. Peptid-Scanning bietet nicht nur eine einfache Möglichkeit, die Antikörper-Bindungsregion auf der Karte, sondern erleichtert auch Aminosäure (aa) Mutagenese durch Rückstands Scannen oder Substitution die Bindungswechselwirkung zwischen jedem aa-Rest des Epitop-Peptid und die CDRs des Antikörpers zu bewerten. <p class = &quot;jove_content&quot;&gt; Hier beschreibt die vorliegende Studie ein Protokoll für die effiziente Identifikation des linearen Epitop des gelben Grouper nervös Nekrose - Virus (YGNNV) Hüllprotein mit einem neutralisierenden mAb 2, 3, 4. Das Protokoll enthält mAb Vorbereitung, Konstruktion und Expression von seriell verkürzten rekombinanten Proteine, synthetische überlappende Peptid Design, Dot-Blot-Hybridisierung, Alanin-Scanning und Substitutionsmutagenese. die hohen Kosten für die Peptidsynthese unter Berücksichtigung, wurde der Schritt des seriell die rekombinanten Proteine ​​eines gewünschten Zielproteins Verkürzen modifiziert und die antigene Region wurde auf etwa 100 bis 200 aa Reste, bevor der synthetische Peptidarray Dot-Blot-Analyse durchgeführt wurde eingeengt.

<table><tbody><tr><td>Hybrid-SFM medium</td><td>Gibco</td><td>12045-076</td><td></td></tr><tr><td>Dulbeccos&#039;s Phophate-Buffered Saline (PBS)</td><td>Gibco</td><td>21600-069</td><td></td></tr><tr><td>Pfu DNA Polymerase&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>EP0502&amp;nbsp;</td><td>Including buffers</td></tr><tr><td>T4 DNA Ligase</td><td>Roche</td><td>10799009001</td><td>Including buffers</td></tr><tr><td>NdeI&amp;nbsp;</td><td>New England Biolabs</td><td>R0111S</td><td>Including buffers</td></tr><tr><td>XhoI&amp;nbsp;</td><td>New England Biolabs</td><td>R0146S</td><td>Including buffers</td></tr><tr><td>pET-20b(+) vector</td><td>Novagen, Merck Millipore</td><td>69739</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; DH-5&amp;alpha; competent cell</td><td>RBC Bioscience</td><td>RH617</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL-21(DE3) competent cell</td><td>RBC Bioscience</td><td>RH217</td><td></td></tr><tr><td>Ampicillin</td><td>Amresco</td><td>0339-25G</td><td></td></tr><tr><td>LB broth&amp;nbsp;</td><td>Invitrongen</td><td>12780-052</td><td></td></tr><tr><td>Isopropylthio-&amp;beta;-D-thiogalactoside (IPTG)</td><td>MDBio, Inc.</td><td>101-367-93-1</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td><td>J.T.Baker</td><td>X251-07</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Glycine</td><td>Amresco</td><td>0167-5KG</td><td></td></tr><tr><td>Tris</td><td>Affymetrix, USB</td><td>75825</td><td></td></tr><tr><td>NaCl</td><td>Amresco</td><td>0241-1KG</td><td></td></tr><tr><td>EDTA</td><td>Amresco</td><td>0105-1KG</td><td></td></tr><tr><td>Glycerol</td><td>Amresco</td><td>0854-1L</td><td></td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S2002-500G</td><td></td></tr><tr><td>BCIP/NBT</td><td>PerkinElmer</td><td>NEL937001PK</td><td></td></tr><tr><td>Goat Anti-Mouse IgG, Fc fragment antibody</td><td>Jackson ImmunoResearch</td><td>115-055-008</td><td></td></tr><tr><td>Immobilon-P (Polyvinylidene fluoride, PVDF)</td><td>Merck Millipore</td><td>IPVH00010</td><td></td></tr><tr><td>Protein G Agarose Fast Flow</td><td>Merck Millipore</td><td>16-266</td><td></td></tr><tr><td>QIAquick PCR Purification kit</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>UVP BioSpectrum 600 Image System</td><td>UVP</td><td>n/a</td><td></td></tr><tr><td>VisionWorks LS Analysis Software Ver 6.8</td><td>UVP</td><td>n/a</td><td></td></tr><tr><td>MyCycler thermal cycler</td><td>BioRad</td><td>1709713</td><td></td></tr></tbody></table>

peptide scanning-assisted identification of a monoclonal antibody-recognized linear b-cell epitope

Die Identifizierung eines antigenes Epitop von dem Immunsystem ermöglicht das Verständnis des Schutzmechanismus von neutralisierenden Antikörpern, die die Entwicklung von Impfstoffen und Peptid-Medikamente können erleichtern. Peptid-Scanning ist eine einfache und effiziente Methode, die ohne weiteres das lineare Epitop durch einen monoklonalen Antikörper (mAb) erkannt abbildet. Hier präsentieren die Autoren ein Epitop Bestimmung Methodik seriell verkürzten rekombinanten Proteinen, synthetischen Peptid-Design und Dot-Blot-Hybridisierung für die Antigen-Erkennung von Nerven Nekrose-Virus-Hüllprotein mit einem neutralisierenden mAb beteiligt sind. Diese Technik beruht auf dem Dot-Blot-Hybridisierung von synthetischen Peptiden und mAbs auf einem Polyvinylidenfluorid (PVDF) -Membran. Die minimale antigen Bereich eines viralen Hüllprotein vom RG-M56 mAb erkannt durch die Schritt-für-Schritt-getrimmten Peptidkartierung auf ein 6-mer-Peptid-Epitops eingegrenzt werden. Zusätzlich Alanin-Scanning-Mutagenese und Rückstands substitution kann die Bindungs ​​Bedeutung jeder Aminosäurerest, aus denen das Epitop charakterisieren, durchgeführt werden. Die Reste, die die Epitop-Website flankieren, wurden gefunden kritische Rollen in Peptidkonformation Regulation zu spielen. Das identifizierte Epitop-Peptid kann verwendet werden, um Kristalle von Epitop-Peptid-Antikörper-Komplexe für eine Röntgenbeugungsuntersuchung zu bilden und funktionellen Wettbewerb oder für Therapeutika.

Das Ziel dieses Experimentes war es ein Epitop durch Dot-Blotting unter Verwendung von mAb zu identifizieren. Um schnell und effizient verengen die antigene Region anerkannt durch mAb, die in voller Länge und seriell abgeschnitten YGNNV rekombinanten Hüllproteine mit einem 6xHis Fusions - Tag am C-Terminus wurden aus einem E. coli - PET - Expressionssystem 10 (1A) ausgedrückt. Die resultierenden rekombinanten Proteine ​​wurden auf ...

Watch this Scientific Journal Video about Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope at JoVE.com

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Peptid-Scanning-gestützte Identifizierung eines monoklonalen Antikörpers anerkannte Linear B-Zell-Epitop

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

peptide-scanning-assisted-identification-monoclonal-antibody

Research

JoVE Journal

Immunology and Infection

9.3K Aufrufe.  Academia Sinica. Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Serie: Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Halbautomatische Biopanning bakterielle Anzeige Bibliotheken für Peptid Affinität Reagenz Entdeckung und Analyse der daraus resultierenden Isolate

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Forschung

Biochemie

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

Personalisierte Peptidarrays für die Erkennung von HLA Alloantibodies in Organtransplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

Peptid: MHC-Tetramer-basierte Anreicherung von Epitop-spezifischer T-Zellen

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Immunologie und Infektion

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein.

Überlappende Peptid-Bibliothek zuordnen Qa-1 Epitope in einem Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Generation von diskriminierendem menschlichen monoklonalen Antikörpern aus seltenen Antigen-spezifische B-Zellen im Blut zirkulierenden

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

Biotechnik

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

Einzellige Screening-Methode zur Auswahl und Wiederherstellung von Antikörpern mit gewünschten Besonderheiten aus angereicherten menschlichen Gedächtnis B-Zellpopulationen

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct functional subsets based on the cytokines they secreted: for example, IFN-&#947; for CD4 T helper 1 cells, IL-4 and IL-13 for CD4 T helper 2 cells, IL-21 for CD4 T follicular helper cells, and IL-17 for CD4 T helper 17 cells. Analysis of hepatitis B virus (HBV)-specific CD4 T cells based on cytokine secretion after HBV-derived peptides stimulation could provide information not only about the magnitude of HBV-specific CD4 T-cell response but also about the functional subsets of HBV-specific CD4 T cells. Novel approaches, such as transcriptomics and metabolomics analysis, could provide more detailed functional information about HBV-specific CD4 T cells. These approaches usually require isolation of viable HBV-specific CD4 T cells based on peptide-major histocompatibility complex-II multimers, while currently the information about HBV-specific CD4 T-cell epitopes is limited. Based on an HBV-derived peptide matrix, a method has been developed to evaluate HBV-specific CD4 T-cell responses and identify HBV-specific CD4 T-cell epitopes simultaneously using peripheral blood mononuclear cells samples from chronic HBV infection patients.

Based on a hepatitis B virus (HBV)-derived peptide matrix, HBV-specific CD4 T-cell responses could be evaluated in parallel with identification of HBV-specific CD4 T-cell epitopes.

Analyse von HBV-spezifischen CD4-T-Zell-Reaktionen und Identifizierung von HLA-DR-beschränkten CD4-T-Zell-Epitopen auf Basis einer Peptidmatrix

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct ...

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 ...

Biologie

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.