Ringraziamo il Dr. James Robinson per il suo generoso dono della A32, 17b, 48d, e C11 anticorpi monoclonali. PGT 121 è stato ottenuto attraverso il Programma reagente NIH AIDS, Divisione di AIDS, NIAID, NIH (Cat # 12343). Questo lavoro è stato sostenuto da una Fondazione canadese per l&#39;innovazione programma Leader # 29866, da un esercizio CIHR # 257792, da un FRQS Istituzione di Young Scientist concede # 24639 di AF e da un finanziamento continuo CRCHUM nonché da un CIHR sovvenzione catalizzatore # 126630 AF e MR. AF è il destinatario di un FRSQ Chercheur Boursier Junior 1 borsa di studio # 24639. MV è stato sostenuto da un CIHR Dottorato Research Award # 291485.

1 Giorno 1 - Colture Cellulari <ol><li> Piastra 2 x 10 4 osteosarcoma umano (HOS) cellule per pozzetto in una, 96 pozzetti coltura cellulare opaco adatto per la lettura luminescenza. Utilizzare Dulbecco Modified Eagle Medium (DMEM) supplementato con 10% di siero fetale bovino (FBS) e 100 U / ml di penicillina-streptomicina. Incubare fino al giorno successivo a 37 ° C, 5% di CO 2. </li></ol> 2 Giorno 2 - polietilenimmina (PEI) Transfezione <ol><li> Preparare mix trasfezione in base alle fasi successive. Regolare reagenti e le quantità di DNA secondo il numero di pozzi che devono essere trasfettate con la stessa Env. </li><li> Tubo A: Aggiungi 10 ng plasmide Tat-codifica (ad esempio PTAT-III, 27) e 150 ng Env-plasmide codificante per 5 microlitri DMEM supplementato con 25 HEPES mM. </li><li> Il plasmide Tat-codifica è necessaria solo se si utilizza Tat-dipendente Env-plasmidi codificanti, come pSVIII. </li><li> Tubo B: Aggiungere 450 ng PEI (da un 1mg / ml soluzione) a 5 microlitri DMEM. </li><li> Aggiungere il contenuto di tubo B a tubo A. Mescolare accuratamente con il vortex per 10 secondi e incubare mix trasfezione 10 minuti a temperatura ambiente (22 ° C). </li><li> Aggiungere 10 ml di miscela di trasfezione per pozzetto della piastra a 96 pozzetti. Incubare per 48 ore a 37 ° C, 5% di CO 2. </li></ol> 3 Giorno 4 - ELISA <ol><li> Eseguire tutti gli esperimenti a temperatura ambiente per ridurre al minimo possibile l&#39;endocitosi di ENV / anticorpi complessi. </li><li> Preparare 250 ml di tampone di lavaggio per piastra in uso allo stesso tempo. Lavaggio Buffer è 1x salina tamponata con Tris (TBS) pH 7.5 (50 mM Tris-Cl, pH 7.5, 150 mM NaCl), supplementato con 1 mM MgCl 2 e CaCl 1,8 mm 2. </li><li> Preparare 125 ml di tampone di bloccaggio per piastra con l&#39;aggiunta di latte in polvere scremato 1% e 5 mM Tris pH 8.0 a Washing Buffer. </li><li> Rimuovere i supporti di coltura cellulare e mix trasfezione (surnatante) dalla piastra a 96 pozzetti. </li><li> Aggiungere 100 μ; L di tampone di bloccaggio per pozzetto e incubare 20 minuti a temperatura ambiente. </li><li> Rimuovere il surnatante e aggiungere 50 ml di anticorpi (o siero) per bene, diluito alla concentrazione adeguata nel tampone di bloccaggio. Tipicamente, utilizzare una concentrazione di 1 mg / ml. Incubare 1 ora a temperatura ambiente. </li><li> Lavare 3x con 100 microlitri tampone di bloccaggio e quindi ripetere il lavaggio 3x processo con 100 microlitri di lavaggio tampone. </li><li> Rimuovere il surnatante, aggiungere 100 microlitri tampone di bloccaggio, e incubare 5 minuti a temperatura ambiente. </li><li> Rimuovere il surnatante e aggiungere 50 ml di anticorpo secondario, diluito 1/3000 in tampone di bloccaggio. Variare diluizione anticorpo ottimale in base alle differenze produttore. Incubare 40 minuti a temperatura ambiente. </li><li> Lavare 3x con 100 microlitri tampone di bloccaggio e quindi ripetere il lavaggio 3x processo con 100 microlitri di lavaggio tampone. </li></ol> 4 Acquisizione Dati <ol><li> Rimuovere il surnatante dal piatto e aggiungere 30 microlitri 1x chemiluminescenza (ECL) substrato per pozzetto. </li><li> Acquisire chemsegnale iluminescence per 1 sec / bene su un piatto adatto-reader secondo le istruzioni del produttore. Tempo di lettura può variare in base alle differenze di hardware. </li></ol>

<ol>
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Gli autori dichiarano assenza di conflitti di interesse.

Questo test è ottimizzato per rilevare l&#39;interazione di anticorpi monoclonali specifici da HIV-1 trimeric Env espresso sulla superficie cellulare. Una volta che il protocollo è stato stabilito, può essere utilizzato a medio-elevate produzioni con bassi costi di materiale complessivi e piccole quantità di anticorpi. Dal momento che questo saggio è basato trasfezione-, può essere facilmente adattato per coexpression delle proteine ​​cellulari, come CD4 al fine di studiare i loro effetti sulla Env conformazio...

Umana di tipo 1 del virus dell&#39;immunodeficienza (HIV-1) ingresso, mediato dalle trimeriche glicoproteine ​​dell&#39;involucro virale (ENV) è il primo passo del ciclo infettivo. Essendo l&#39;unico antigene virale esposto presentato alla superficie di virioni, la Env trimero suscita anticorpi neutralizzanti e nonneutralizing. Come tale, esso rappresenta un candidato interessante per la progettazione del vaccino immunogeno. Tuttavia, le prove di vaccinazione con Env in forme solubili o ricombinanti hanno suscitato risposte solo con efficacia minima contro la maggior parte primaria da HIV-1 isolati 1-3. Tuttavia, l&#39;efficacia parziale osservato nel trial vaccino RV144 4 rinnovato interesse per HIV-1 Env come candidato immunogeno. Questo è stato confermato da un recente studio che descrive che gli anticorpi vaccino-suscitato anti-Env sono stati sufficienti a generare un certo grado di protezione contro SIV e HIV sfide 5. Dopo essere sintetizzati nel reticolo endoplasmatico, il glycoprote Envin precursori, gp160, subisce varie modificazioni post-traduzionali che sono fondamentali per la sua capacità di alimentare il processo di fusione virale. Il precursore Env deve piegare correttamente e associato in trimeri prima di essere spaccati nelle sue extra-citoplasmatici gp120 e gp41 subunità transmembrana 6-10, con interazioni non covalenti mantenendo il collegamento gp120-gp41. La macchina cellula infetta è anche responsabile pesantemente glicosilazione Env, che comprende circa il 50% della sua massa totale 11,12. La struttura complessa risultante permette Env di essere conformazione flessibile 13,14, mentre fornisce un metastabilità che è pensato per consentire Env di adattarsi e nascondere alcuni epitopi altamente immunogenico che altrimenti sarebbero esposti 15-19, sottolineando l&#39;importanza di comprendere meglio le diverse conformazioni campionata dal Env trimero nativo. Ad oggi, sono state sviluppate diverse tecniche e con successo utilizzati per studiare Env ch conformazionaleanges. Tuttavia, essi variano nelle loro limitazioni, essendo spesso limitato a specifici contesti Env. Ad esempio, risonanza plasmonica di superficie o immunoprecipitazione con anticorpi monoclonali specifici di conformazione (MAK, MAB), si basano sia su monomeriche molecole solubili o solubilizzati Env che sono noti per essere immunogenetically diverso dal loro forme trimeriche 20,21. Recenti studi suggeriscono anche che la scissione colpisce conformazioni Env conseguente l&#39;esposizione di epitopi principalmente riconosciuti da anticorpi nonneutralizing 14,22,23. Qui si descrive in dettaglio un metodo che permette di determinare facile e veloce della conformazione del-cellularmente espresso Env trimeri 18,24-26. A seguito di trasfezione transiente di Env in una linea di cellule aderenti umano il legame di anticorpi specifici Env-viene rilevato utilizzando una semplice reazione di chemiluminescenza. Questa tecnica può anche essere utilizzato per caratterizzare la preferenza conformazionale di conformazione Depenanticorpi dent. Così, questo saggio fornisce un metodo di rilevazione robusta e altamente flessibile.

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			<td height="42" style="height:42px;width:216px;">White Opaque Tissue Culture Plate, 96 well, Flat Bottom</td>
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conformational evaluation of hiv-1 trimeric envelope glycoproteins using a cell-based elisa assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Utilizzando la procedura generale di cui sopra, abbiamo adattato il protocollo per saggiare l&#39;impatto dei CD4 solubile (sCD4) e CD4 cellulare coexpressed sull&#39;esposizione di epitopi CD4i su entrambi wild-type (WT) o mutato Env, come descritto in precedenza 18,24, 25,28. figura 1 rappresenta schematicamente la procedura generale e l&#39;esposizione di epitopi CD4i seguito di trattamento con sCD4 o da coexpression di CD4 cellulare 18. Nella figura 2, abbiamo ...

Watch this Scientific Journal Video about Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay at JoVE.com

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

Valutazione conformazionale di HIV-1 trimeric busta glicoproteine ​​Utilizzando una base di Cell-ELISA Saggio

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

conformational-evaluation-hiv-1-trimeric-envelope-glycoproteins-using

Research

JoVE Journal

Immunology and Infection

14.2K Views.  Université de Montréal. Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

Valutazione conformazionale di HIV-1 trimeric busta glicoproteine ​​Utilizzando una base di Cell-ELISA Saggio

Valutazione conformazionale di HIV-1 trimeric busta glicoproteine Utilizzando una base di Cell-ELISA Saggio