Questo lavoro è stato sostenuto da sovvenzioni NIH R01-GM-098977 e R21-AI-098.122 per CBK e KEG. RSS è stato sostenuto in parte da una Luce Foundation Fellowship e in parte da una borsa di studio Thayer Innovation Program presso la Scuola di Ingegneria Thayer.

Quattro attività principali comprendono il saggio di legame peptide-MHC II: 1-Rilegatura: peptidi di prova in concorrenza con peptidi marcati di controllo per l&#39;associazione in soluzione di proteine ​​MHC II solubili. Binding è misurata su una vasta gamma di concentrazioni di peptide di prova. 2-Capture: Dopo la reazione di legame avvicina equilibrio, complessi peptide-MHC II vengono catturati e separati dal peptide legato e proteine ​​mediante riconoscimento dipendente dalla conformazione con un anticorpo immobilizzato. 3-Detection: Catturato peptide di controllo è quantitativamente rilevata tramite fluorescenza in tempo risolto. 4-Analisi: i dati spettroscopici sono trattati, tracciati e analizzati per accertare dose-dipendenti proprietà di legame di prova peptidi e le proteine ​​MHC II. In vari passaggi della procedura sotto, l&#39;uso di un robot liquido gestione è consigliato se uno è disponibile. Soprattutto in un formato 384-bene, erogazione automatizzata e diluizione dei liquidi minimizza errori degli utenti, risultati in piùpozzo alla ben consistenti volumi ei rendimenti strette 95% intervalli di confidenza per valori di IC 50 rispetto ai saggi 96 pozzetti manuali (dati non mostrati). Se un robot di gestione dei liquidi non è disponibile, i passaggi annotati con &quot;(manipolazione liquido robot)&quot; può essere fatto a mano. Allo stesso modo, se possibile, si raccomanda un lavatore automatico che è compatibile con il formato 384 pozzetti. Questo standardizza efficacemente il processo di lavaggio piatto. Se una rondella targa non è disponibile, i passaggi annotati con &quot;(lavatore)&quot; può essere fatto a mano. 1. Rivestire la piastra ELISA (Day 1) <ol><li> Diluire L243 anticorpo magazzino (0,5 mg / ml) in tampone borato ad una concentrazione di lavoro di 10 mg / ml. <ol><li> Per rivestire un piatto unico da 384 pozzetti, aggiungere 200 ml di brodo di anticorpi a 9,8 ml di tampone borato. (Vedere calcolatrice foglio supplementare per il ridimensionamento alternativo). </li></ol></li><li> Aggiungere 25 microlitri della soluzione L243 anticorpo a ciascun pozzetto di una 384 well high-binding bianco piastra ELISA. (Manipolazione robot liquido) </li><li> Sigillare la piastra con un film di poliestere e incubare una notte a 4 ° C. Nota: Ogni piastra ELISA a 384 e può ospitare fino a 15 peptidi di prova, a otto diluizioni per un allele MHC II, nonché i controlli positivi e negativi. Questo in ultima analisi produrre misurazioni triplice copia. </li></ol> 2. Effettuare il test Peptide diluizioni (1 ° giorno) <ol><li> Partendo da uno stock 10 mM di ciascun peptide prova in DMSO, effettuare la seguente serie di diluizioni in citrato tampone fosfato utilizzando piastre di polipropilene da 384 pozzetti (Tabella 1). (Manipolazione robot liquido) Nota: Una piastra in polipropilene pieno 384 pozzetti richiede tre piastre ELISA separati. Un terzo di una piastra di polipropilene richiede solo una piastra ELISA (Figura 1). </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody align="center"><tr><td rowspan="2"> Numero di diluizione </td><td> Volume to prendere da prima diluizione / magazzino </td><td> Volume tampone citrato per aggiungere </td><td> Conc. di diluizione </td><td> Conc. in reazione di legame </td><td> Conc. in neutralizzato ELISA </td></tr><tr><td> (Microlitri) </td><td> (Microlitri) </td><td> (Micron) </td><td> (Micron) </td><td> (Micron) </td></tr><tr><td> 1 </td><td> 0.7 </td><td> 35.1 </td><td> 195,531 </td><td> 97,765 </td><td> 48,883 </td></tr><tr><td> 2 </td><td> 14.3 </td><td> 14.3 </td><td> 97,765 </td><td> 48,883 </td><td> 24,441 </td></tr><tr><td> 3 </td><td> 7.1 </td><td> 28.7 </td><td> 19,389 </td><td> 9,695 </td><td> 4,847 </td></tr><tr><td> 4 </td><td> 14.3 </td><td> 14.3 </td><td> 9,695 </td><td> 4,847 </td><td> 2.424 </td></tr><tr><td> 5 </td><td> 7.1 </td><td> 28.7 </td><td> 1.923 </td><td> 0,961 </td><td> 0,481 </td></tr><tr><td> 6 </td><td> 14.3 </td><td> 14.3 </td><td> 0,961 </td><td> 0,481 </td><td> 0.24 </td></tr><tr><td> 7 </td><td> 7.1 </td><td> 28.7 </td><td> 0,191 </td><td> 0.095 </td><td> 0.048 </td></tr><tr><td> 8 </td><td> 14.3 </td><td> 14.3 </td><td> 0.095 </td><td> 0.048 </td><td> 0.024 </td></tr><tr><td colspan="6"> Rimuovere e gettare 7.1 ml di soluzione di peptide dalla diluizione numero 8. </td></tr></tbody></table> Tabella 1. Preparazione di prova serie di diluizioni peptide. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51308/51308fig1highres.jpg" src="/files/ftp_upload/51308/51308fig1.jpg" /> Figura 1. Piastra Mappa del Peptide Binding Assay. (A) Mappa delle diluizioni del peptide in polipropilene 384 pozzetti. Ogni lastra in polipropilene in grado di ospitare tre gruppi di 15 peptidi in concorrenza reazioni di legame nei confronti di un cantare<html> allele le MHC II. (B) Dopo la reazione di legame avvicina equilibrio, ciascun gruppo peptide viene trasferito, in triplice copia, ad una piastra a 384 pozzetti ELISA separato che è stato prerivestita con anticorpo anti-MHC II. 3. Preparare il MHC II Master Mix (Day 1) <ol><li> Rendere il Reaction Buffer <ol><li> Aggiungere 80 ml di 1 mM Pefa Bloc e 60 mg di ottile-β-D-glucopyranaside a 3.920 ml di citrato tampone fosfato. (Vedere calcolatrice foglio supplementare per il ridimensionamento alternativo). </li></ol></li><li> Diluire le soluzioni di archivi MHC II selezionati a 101 nm in Reaction Buffer utilizzando un tubo da 15 ml (Tabella 2). Nota: concentrazione MHC II alla fine sarà 50 nM nella reazione di legame e 25 nM nel saggio ELISA neutralizzato. Le concentrazioni di archivi MHC II variano a seconda del numero di produttori lotto. (Vedere calcolatrice foglio supplementare). </li></ol>&quot;&gt; <tbody><tr><td> MHC II alleli DRB1 *: </td><td> 1501 </td></tr><tr><td> MHC II Stock Concentrazione (mg / ml) </td><td> 1.3 </td></tr><tr><td> MHC II Stock Concentration (mm) </td><td> 20 </td></tr><tr><td> Vol. II MHC Archivio per aggiungere (ml) </td><td> 14.65 </td></tr><tr><td> Vol. Reaction Buffer per aggiungere (ml): </td><td> 2.885,35 </td></tr><tr><td> MHC II Master Mix Concentration (nm) </td><td> 101 </td></tr></tbody></table> Tabella 2. Preparazione del master mix MHC II. 4. Preparare i controlli negativi e positivi (1 ° giorno) <ol><li> Aggiungere 21.5 ml di citrato tampone fosfato al pozzo &quot;controllo negativo&quot; della piastra in polipropilene 384 pozzetti dal punto 2.1 (Figura 1A). </li><li> Aggiungere 21.5 ml di citrato tampone fosfato al pozzo &quot;controllo positivo&quot; della piastra in polipropilene da 384 pozzetti. Nota: la &quot;posizionecontrollo effettivo &quot;sarà completato nella sezione 5, in basso. </li><li> Rimuovere 49.5 ml di MHC II Master Mix dal punto 3.2 e pipetta in una provetta da 1,5 ml Eppendorf. </li><li> Aggiungere 0,5 ml di tampone citrato alle 49.5 ml di MHC II Master Mix dal punto 4.3. </li><li> Aggiungere 21.5 ml di concentrazione adeguati MHC II Master Mix dal punto 4.4 al pozzo &quot;controllo negativo&quot; della piastra in polipropilene 384 pozzetti (Figura 1A). Nota: questo esempio rappresenta il controllo negativo zero segnale contenente proteine ​​MHC II, NO peptide di controllo, e NO peptide test. </li></ol> 5. Aggiungere il comando appropriato Peptide al MHC II Master Mix (Day 1) <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> MHC II alleli DRB1 * </td><td> Biotinilato controllo Peptide </td><td> (Residui) </td><td> Sequenza </td></tr><tr><td> 0101 </td><td> Flu-HA-B </td><td> (306-318) </td><td> Biotina-(Ahx) (Ahx) PRYVKQNTLKLAT-ammide </td></tr><tr><td> 0301 </td><td> Mioglobina </td><td> (137-148) </td><td> Biotina-(Ahx) - (Ahx)-LFRKDIAAKYKE-OH </td></tr><tr><td> 0401 </td><td> YAR-B </td><td> (1-14) </td><td> Biotina-(Ahx) (Ahx) YARFQSQTTLKQKT-OH </td></tr><tr><td> 0701 </td><td> TetTox-B </td><td> (830-843) </td><td> Biotina-(Ahx) (Ahx) QYIKANSKFIGITE-OH </td></tr><tr><td> 1101 </td><td> Flu-HA-B </td><td> (306-318) </td><td> Biotina-(Ahx) (Ahx) PRYVKQNTLKLAT-amide </td></tr><tr><td> 1501 </td><td> MBP-B </td><td> (84-102) </td><td> Biotina-(Ahx) (Ahx) NPVVHFFKNIVTPRTPPPS-OH </td></tr></tbody></table> * Si consiglia di ordinare 10 mg scala per l&#39;economia. Tabella 3. Controllo Peptidi per varie MHC II alleli. * <ol><li> Diluire il peptide di controllo (conservato a 400 micron di DMSO) 01:20 in DMSO fresca per produrre uno stock diluita a 20 micron. <ol><li> Aggiungere 25 ml 400 control micron peptide475 microlitri DMSO. Nota: questo è un grande eccesso, ma permette la gestione accurata di soluzioni viscose DMSO. </li></ol></li><li> Diluire ulteriormente il controllo Peptide dal punto 5.1 (20 mm) 1:100 in MHC II Master Mix dal punto 3.2. <ol><li> Aggiungere 28,8 ml di controllo Peptide diluito nel passaggio 5.2 per i restanti 2,850.5 ml di MHC II Master Mix. </li></ol></li><li> Aggiungere 21,5 ml di MHC II Master Mix con peptide di controllo (passo 5.2) al pozzetto di controllo positivo della piastra polipropilene 384 pozzetti (passo 4.2) (Figura 1A). Nota: questo esempio rappresenta il controllo positivo alto segnale contenente proteine ​​MHC II, controllo peptide, e NO peptide test. </li></ol> 6. Effettuare la reazione di legame (Day 1) <ol><li> Aggiungere il MHC II Master Mix contenente controllo Peptide (passo 5.2) a ciascuna delle diluizioni test Peptide (passo 2.1) in un rapporto 1:1 a creare la reazione di legame. <ol><li> Aggiungere 21.5 ml di MHC II Master Mix contenenti Concontrollo Peptide dal punto 5,2-21,5 ml di ogni diluizione di prova Peptide dal punto 2.1. (Manipolazione robot liquido) </li></ol></li><li> Sigillare la reazione di legame con un film di poliestere e incubare 12-24 ore in un incubatore non-CO 2 controllata a 37 ° C senza agitazione. </li></ol> 7. Neutralizzare e Trasferire la reazione di legame (Day 2) <ol><li> Rimuovere la piastra in polipropilene 384 pozzetti contenente la reazione di legame (punto 6.2) dal termostato 37 ° C. </li><li> Diluire 1:1 reazione di legame con la neutralizzazione Buffer. (Manipolazione robot liquido) <ol><li> Aggiungere 43 ml di tampone di neutralizzazione di ogni reazione di legame bene. </li></ol></li><li> Rimuovere la piastra ELISA (passo 1.3) da 4 ° C e lavare con 3x 60 ml / pozzetto di PBS-0.05% Tween 20. (Lavatore) </li><li> Trasferire 25 microlitri di ogni reazione di legame neutralizzato dal punto 7.2 in pozzetti in triplicato del 384 pozzetti ELISA anticorpo piastra rivestita dal punto 7.3. (Figura 1 </strong&gt;) (manipolazione robot liquido) </li><li> Coprire la piastra ELISA con un film di poliestere e posto in un incubatore a 37 ° C per 2,5 ore o in un frigorifero a 4 ° C durante la notte. </li></ol> 8. ELISA Development (Day 2 o 3) <ol><li> Rimuovere la piastra ELISA dal punto 7.5, lavare 3 volte con 60 microlitri / pozzetto di PBS-0.05% Tween. (Lavatore) </li><li> Diluire la soluzione streptavidina-Europium magazzino (0,1 mg / ml streptavidina, 7 Eu 3 + / streptavidina) 1,000 volte nel tampone del saggio DELFIA. <ol><li> Per una piastra da 384 pozzetti, diluire 10 ml streptavidina-Europium in 10 ml di tampone del saggio. </li></ol></li><li> Aggiungere 25 ml di diluito streptavidina-europio a ciascun pozzetto della piastra a 384 pozzetti ELISA dal punto 8.1. (Manipolazione robot liquido) </li><li> Coprire la piastra con un film di poliestere e posto al buio a temperatura ambiente per 1 ora. Nota: Durante l&#39;incubazione 1 ora, rimuovere la soluzione Enhancement dal frigorifero e mettere da parte 10 ml / piastra al buio a rotemperatura om. </li><li> Seguendo la incubazione di 1 ora, lavare la piastra a 384 pozzetti ELISA dal punto 8.3 3x con 60 pl / pozzetto di PBS-Tween 0,05%. (Lavatore) </li><li> Aggiungere 25 ml di soluzione Enhancement a ciascun pozzetto della piastra a 384 pozzetti ELISA dal punto 8.5. (Manipolazione robot liquido) </li><li> Coprire la piastra ELISA 384 pozzetti con un film di poliestere e lasciare che la piastra di sedersi al buio a temperatura ambiente per 10-15 min. </li><li> A seguito del 10-15 min di incubazione, leggere la fluorescenza della piastra a 384 pozzetti ELISA dal punto 8.7 utilizzando un lettore di tempo risolto fluorescente piastra con impostazioni europio (per esempio, Inizio Int: 200, Stop Int:. 1.000, Ex 340, Em . 615, Cutoff: Nessuno, PMT: Auto, Letture / Well: 50). </li></ol> 9. Analisi dei dati <ol><li> Inserire i dati in formato grafico XY con 3 valori ripetute nelle colonne side-by-side (X = concentrazione di peptide prova; Y = misurazione della fluorescenza). </li><li> Log trasformare i valori X per tutti i dati: X = log (X) </li><li> Montare i TRA registrodati nsformed con un sito di legame modello competitivo per estrarre il valore di IC 50. </li><li> Vincolare il valore inferiore al valore di controllo negativo medio. </li><li> Globalmente adattare il valore superiore e assicurare che il parametro di misura globale è inferiore o uguale al controllo positivo. </li></ol>

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E., Bailey-Kellogg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-guided+deimmunization+of+therapeutic+proteins.">Structure-guided deimmunization of therapeutic proteins.</a> J. Comput. Biol. 20, 152-165 (2013).</li><li>Parker, A. S., Griswold, K. E., Bailey-Kellogg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+therapeutic+proteins+to+delete+T-cell+epitopes+while+maintaining+beneficial+residue+interactions.">Optimization of therapeutic proteins to delete T-cell epitopes while maintaining beneficial residue interactions.</a> J. Bioinform. Comput. Biol. 9, 207-229 (2011).</li><li>Parker, A. S., Zheng, W., Griswold, K. E., Bailey-Kellogg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+algorithms+for+functional+deimmunization+of+therapeutic+proteins.">Optimization algorithms for functional deimmunization of therapeutic proteins.</a> BMC Bioinformatics. 11, 180 (2010).</li><li>De Groot, A. S., Martin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+risk,+improving+outcomes:+Bioengineering+less+immunogenic+protein+therapeutics.">Reducing risk, improving outcomes: Bioengineering less immunogenic protein therapeutics.</a> Clin. Immunol. 131, 189-201 (2009).</li><li>Wang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+assessment+of+MHC+class+II+peptide+binding+predictions+and+evaluation+of+a+consensus+approach.">A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach.</a> PLoS Comput. Biol. 4, e1000048 (2008).</li><li>Sette, A., Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+vaccinology:+developing+vaccines+in+the+era+of+genomics.">Reverse vaccinology: developing vaccines in the era of genomics.</a> Immunity. 33, 530-541 (2010).</li><li>Moise, L., Cousens, L., Fueyo, J., De Groot, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+genomics+and+immunoinformatics+to+produce+improved+vaccines.">Harnessing the power of genomics and immunoinformatics to produce improved vaccines.</a> Expert Opin. Drug Discov. 6, 9-15 (2011).</li><li>Sturniolo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+tissue-specific+and+promiscuous+HLA+ligand+databases+using+DNA+microarrays+and+virtual+HLA+class+II+matrices.">Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices.</a> Nat. Biotechnol. 17, 555-561 (1999).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologic+drugs+set+to+top+2012+sales.">Biologic drugs set to top 2012 sales.</a> Nat. Med. 18, 636 (2012).</li><li>Seib, K. L., Zhao, X., Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+vaccines+in+the+era+of+genomics:+a+decade+of+reverse+vaccinology.">Developing vaccines in the era of genomics: a decade of reverse vaccinology.</a> Clin. Microbiol. Infect. 18, 109-116 (2012).</li><li>Jiang, W., Boder, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+engineering+and+analysis+of+peptide+binding+to+class+II+MHC.">High-throughput engineering and analysis of peptide binding to class II MHC.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 13258-13263 (2010).</li><li>Justesen, S., Harndahl, M., Lamberth, K., Nielsen, L. -. L. B., Buus, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recombinant+MHC+class+II+molecules+and+high-throughput+peptide-binding+assays.">Functional recombinant MHC class II molecules and high-throughput peptide-binding assays.</a> Immun. 5, 1-20 (2009).</li></ol>

Leonard Moise è impiegato da e detiene stock option in EpiVax, Inc., una società di biotecnologia di proprietà privata si trova a Providence, RI. Il lavoro contenute in questo rapporto di ricerca è libera da ogni pregiudizio che potrebbe essere associato con gli obiettivi commerciali dell&#39;azienda.

Biotherapeutics si sono affermati come una pietra miliare della medicina moderna, che rappresentano quattro delle prime cinque farmaci più venduti nel 2012 32. Il settore biofarmaceutica ha mostrato una crescita sostenuta per diversi anni 6, e il continuo sviluppo di nuovi agenti, così come l&#39;emergere dei biosimilari ha ampliato condotte biofarmaceutiche. Guardando al futuro, valutare e mitigare l&#39;immunogenicità di terapie proteiche diventerà parte integrante dello sviluppo biotherapeut...

An erratum was issued for: <a href="https://www.jove.com/video/51308/a-high-throughput-mhc-ii-binding-assay-for-quantitative-analysis">A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes</a>.&#160; There were typos in the Protocol section and Table 2.
Step 3.1.1 in the Protocol was updated from:
Add 80 &#181;l of 1 mM Pefa Bloc and 60 mg of octyl-&#946;-D-glucopyranaside to 3,920 &#181;l of Citrate Phosphate Buffer. (See supplemental spreadsheet calculator for alternative scaling).
to:
Add 80 &#181;l of 1 mM Pefa Bloc and 60 &#181;g of octyl-&#946;-D-glucopyranaside to 3,920 &#181;l of Citrate Phosphate Buffer. (See supplemental spreadsheet calculator for alternative scaling).
Step 5.2&#160;in the Protocol was updated from:
Further dilute the Control Peptide from step 5.1 (20 mM) 1:100 into the MHC II Master Mix from step 3.2.
to:
Further dilute the Control Peptide from step 5.1 (20 &#181;M) 1:100 into the MHC II Master Mix from step 3.2.
Table 2 was updated from:
<table border="0" cellpadding="0" cellspacing="0" width="376">
	<tbody>
		<tr>
			<td>MHC II Allele DRB1*:</td>
			<td>1501</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (mg/ml)</td>
			<td>1.3</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (mM)</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Vol. MHC II Stock to Add (ml)</td>
			<td>14.65</td>
		</tr>
		<tr>
			<td>Vol. Reaction Buffer to Add (ml):</td>
			<td>2885.35</td>
		</tr>
		<tr>
			<td>MHC II Master Mix Concentration (nM)</td>
			<td>101</td>
		</tr>
	</tbody>
</table>
to:
<table border="0" cellpadding="0" cellspacing="0" width="376">
	<tbody>
		<tr>
			<td>MHC II Allele DRB1*:</td>
			<td>1501</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (mg/ml)</td>
			<td>1.3</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (&#956;M)</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Vol. MHC II Stock to Add (&#956;l)</td>
			<td>14.65</td>
		</tr>
		<tr>
			<td>Vol. Reaction Buffer to Add (&#956;l):</td>
			<td>2885.35</td>
		</tr>
		<tr>
			<td>MHC II Master Mix Concentration (nM)</td>
			<td>101</td>
		</tr>
	</tbody>
</table>

An erratum was issued for: <a href="https://www.jove.com/video/51308/a-high-throughput-mhc-ii-binding-assay-for-quantitative-analysis">A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes</a>.&#160; There were typos in the Protocol section and Table 2.

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

Le proteine ​​sono la classe più rapida crescita di agenti terapeutici 6, e la rapida espansione delle condotte biotherapeutic ha focalizzato sempre più l&#39;attenzione sulle sfide connesse con lo sviluppo e l&#39;uso di farmaci proteici. Una considerazione unica deriva dal fatto che, in un sistema immunitario sano e funzionante, tutte le proteine ​​extracellulari sono campionati dalle cellule presentanti l&#39;antigene (APC). Una volta internalizzato da APC, una proteina viene scissa in piccoli frammenti peptidici, e segmenti immunogenici putativi sono caricati nella scanalatura di classe II principali proteine ​​complessi di istocompatibilità (MHC II). I complessi peptide-MHC II vengono quindi visualizzati sulla superficie APC, e vere peptidi immunogeni, chiamati epitopi delle cellule T, forma ternaria complessi recettore delle cellule MHC II-peptide-T con recettori di superficie delle cellule T CD4 affini 7. Questo evento critico riconoscimento molecolare inizia una cascata di segnalazione complessa, che comporta l&#39;attivazione delle cellule T, il rilascio di citochines, CD4 T cellulo-mediata B maturazione delle cellule, e, infine, la produzione di anticorpi IgG circolanti che si legano e azzera la proteina esogena incriminato. Così, proteine ​​immunogeniche potrebbero essere deimmunized individuando costitutivi epitopi delle cellule T e mutando residui chiave responsabili MHC II formazione del complesso. È notato, comunque, che gli epitopi di cellule T possono essere numerosi e ampiamente distribuiti in tutta proteine ​​immunogeniche, e la maggior parte delle mutazioni-epitopo eliminazione sono suscettibili di provocare una perdita involontaria di una funzione proteica o la stabilità. Pertanto, ingegneria deimmunized bioterapie può essere un obiettivo complesso e tecnicamente impegnativo, ma esistono diversi esempi di successo di T epitopi delle cellule progetti soppressione 3,5,8-12. Diversamente basato sovrainnesti &quot;umanizzazione&quot;, che è in gran parte limitato a terapeutica dell&#39;anticorpo, soppressione epitopo può essere applicato a qualsiasi sostanza bersaglio proteina indipendentemente sequenza, struttura, funzione, o la disponibilità di homologoci ponteggi umani. Il primo passo per l&#39;attuazione di un tale approccio è l&#39;identificazione di epitopi peptidici chiave incorporati all&#39;interno della sequenza della proteina bersaglio. Ad alto rendimento saggi biochimici utilizzando peptidi sintetici e di molecole ricombinanti umani MHC II grado di fornire rapide intuizioni preliminari nella identificazione degli epitopi e mitigazione 1,3-5. Questi saggi tipo ELISA può essere un potente complemento ad altri di progettazione e sviluppo di strumenti di proteine ​​/ vaccino. Per esempio, un approccio sperimentale consolidata di mappatura di epitopi si basa sul tempo, lavoro e notevoli risorse ex vivo saggi di proliferazione cellulare 15. In breve, la sequenza primaria di una proteina bersaglio viene prima divisa in un pannello di peptidi sovrapposti, spesso 15-meri con 12 residui sovrappongono tra peptidi adiacenti. Il pannello peptide è sintetizzato chimicamente e l&#39;immunogenicità di ciascun peptide è testato in uno dei vari test immunologici diversi che impiegano Bloo perifericocellule d mononucleate (PBMC) isolate da donatori umani 13,14. Per garantire una maggiore fiducia nei risultati, i peptidi sono in genere testati in replicato con PBMC da 50 o più donatori diversi. Nei casi in cui deimmunization è l&#39;obiettivo finale, il lavoro è aggravato ulteriormente la necessità di produrre pannelli supplementari di peptidi mutati e collaudare i nuovi pannelli peptidici in saggi PBMC prima di introdurre eventuali mutazioni deimmunizing nella proteina intera lunghezza per la successiva analisi funzionale 10. Mentre questi saggi cellulari rimangono il gold standard per valutare il potenziale immunogenico in pazienti umani, l&#39;efficienza di un approccio esaustivo potrebbe essere migliorata prefiltraggio epitopi immunogeni putativi usando una rapida ed elevata produttività MHC II-peptide saggio di legame. Analogamente, biochimici saggi di legame peptide-MHC II possono essere combinati con predittiva metodi in silico per accelerare radicalmente il processo di identificazione epitopo.Esistono una varietà di strumenti di calcolo per la previsione epitopo T cellulare; esempi includono ProPred 16, MHCPred 17, SVRMHC 18, ARB 19, SMM-align 20, NetMHCIIpan 21, nonché strumenti proprietari come EpiMatrix di EpiVax 22. Allo stesso modo, i predittori epitopi sono stati recentemente associati ad altre bioinformatica e strumenti di modellazione molecolare per produrre proteine ​​algoritmi deimmunization integrati, progettati per ridurre il rischio che le mutazioni deimmunizing potrebbero sconvolgere la struttura e la funzione delle proteine ​​23-26. Mentre diversi predittori epitopi hanno dimostrato di essere ragionevolmente accurata 27,28, risultati computazionali sempre richiedono validazione sperimentale. Rapid, ad alto rendimento, e convenienti metodi sperimentali sono più adatti come filtro preliminare in silico previsioni epitopi. In modo simile, i predittori epitopi possono guidare la selezione antigene per vaccinolo retromarciaGY 29,30. Ad esempio, i progressi nella bioinformatica hanno dato intere schermate genoma che rapidamente identificare i candidati vaccini in forma di proteine ​​intere o epitopi peptidici estratti da proteomi patogeni. Mentre questa tecnologia consente sta rimodellando scoperta e lo sviluppo di vaccini protettivi, introduce una nuova sfida sotto forma di intractably grandi liste di candidati vaccini immunogenico. Saggi di legame throughput elevato peptide-MHC II possono guidare la selezione degli epitopi quantificando peptide affinità di legame e la promiscuità vincolante tra più alleli MHC II. Come con proteine ​​deimmunization, tali metodi sperimentali sono in ultima analisi necessarie per convalidare la previsione computazionale di porta promettente vaccino. Qui, un saggio di legame peptide-MHC II scalata a 384 pozzetti è descritto. Il protocollo è altamente parallelo e riduce i costi dei reagenti del 75% rispetto al precedentemente descritto 96 pozzetti formati lastra 1,3-5. Utilizzando un unico liquidazioned robot manipolatore, questo metodo consente un ricercatore di analizzare facilmente circa novanta peptidi prova in triplice copia in un range di concentrazioni otto e quattro tipi di alleli MHC II in meno di 48 ore. Questo articolo descrive la configurazione di una piastra da 384 pozzetti ELISA per l&#39;analisi di sette peptidi sperimentali contro un allele MHC II, ma calcolatrici foglio di calcolo sono forniti come materiale supplementare, in modo da scalare facilmente l&#39;esperimento a qualsiasi numero di peptidi e / o MHC desiderati molecole II.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:587px;">Sodium Tetraborate Decahydrate</td>
			<td style="width:357px;">Sigma</td>
			<td style="width:217px;">221732</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Citric Acid</td>
			<td>Sigma</td>
			<td>C1901</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Dibasic Sodium Phosphate</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Trizma HCl</td>
			<td>OmniPur</td>
			<td>9310</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Tween-20</td>
			<td>Sigma</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">100% DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Octyl-&#946;-D-Glucopyranaside</td>
			<td>Fischer</td>
			<td>29836-26-8</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Pefa Bloc SCF</td>
			<td>Roche</td>
			<td>1158591600</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Dulbecco&#8217;s 10X Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14200-166</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">DELFIA Assay Buffer</td>
			<td>Perkin Elmer</td>
			<td>4002-0010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">DELFIA Enhancement Buffer</td>
			<td>Perkin Elmer</td>
			<td>4001-0010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Europium Labelled Streptavidin</td>
			<td>Perkin Elmer</td>
			<td>1244-360</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">L243 anti-HLA-DR antibody</td>
			<td>Biolegend</td>
			<td>307602</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Biotinylated tracer peptides</td>
			<td>21st Century Biochemicals</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Test peptides (1-4mg, 85% purity)</td>
			<td>Genscript</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Purified HLA-DRB1 monomers (non-biotinylated)</td>
			<td>Benaroya Research Institute*</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">384-well white EIA/RIA plate</td>
			<td>Thermo</td>
			<td>460372</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Polypropylene 384-well plate</td>
			<td>Costar</td>
			<td>3656</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Film, AxySeal, 80 &#181;m, ELISA Applicator&#160;</td>
			<td>Axygen Asicentific</td>
			<td>PCR-SP</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">MilliQ Water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Epimotion Liquid Handler (or similar)</td>
			<td>Eppendorf</td>
			<td>5075</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Select TS Plate Washer (or similar)</td>
			<td>BioTek</td>
			<td>405</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">SpectraMax Gemini Microplate Reader (or similar)</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4" height="98" style="height:99px;width:1161px;">*Recombinant human MHC II molecules can be obtained from the Benaroya Tetramer Core Laboratory. See: https://www.benaroyaresearch.org/our-research/core-resources/tetramer-core-laboratory</td>
		</tr>
	</tbody>
</table>

a high throughput mhc ii binding assay for quantitative analysis of peptide epitopes

Saggi biochimici con molecole ricombinanti umani MHC II grado di fornire rapide, approfondimenti quantitativi nella identificazione degli epitopi immunogenico, la cancellazione o il design 1,2. Qui, un saggio di legame peptide-MHC II viene scalata a 384 pozzetti. Il protocollo ridotta riduce i costi dei reagenti del 75% ed è più alta velocità di quanto precedentemente descritto protocolli da 96 pozzetti 1,3-5. In particolare, il disegno sperimentale permette l&#39;analisi robusta e riproducibile fino a 15 peptidi contro un allele MHC II per 384 pozzetti ELISA. Utilizzando un&#39;unica movimentazione robot liquido, questo metodo consente un ricercatore di analizzare circa novanta peptidi prova in triplice copia in un range di concentrazioni otto e quattro tipi di alleli MHC II in meno di 48 ore. Altri lavorano nei campi di proteine ​​deimmunization o la progettazione di vaccini e sviluppo può trovare il protocollo per essere utile nel facilitare il proprio lavoro. In particolare, le istruzioni passo-passo e il formato visivodi Giove dovrebbe consentire ad altri utenti di stabilire rapidamente e facilmente questa metodologia nei propri laboratori.

La sequenza peptidica matura di Enterobacter cloacae P99 beta-lattamasi (BLA) (GenBank ID # X07274.1) è stato analizzato con ProPred 16 per leganti peptidici putativi per MHC II allele DRB1 * 1501 (tabella 4). ProPred identificato 117 peptidi nonamer considerando un obbligatorio residuo P1 ancoraggio (cioè una M, L, I, V, F, Y, o W nella posizione 1, che è richiesto per MHC II vincolante 31). Ad una soglia del 5%, peptidi solo con punteggi superiori o uguali a ...

Watch this Scientific Journal Video about A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes at JoVE.com

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

Saggi biochimici con molecole ricombinanti umani MHC II grado di fornire rapide, approfondimenti quantitativi nella identificazione degli epitopi immunogenico, la cancellazione o il design. Qui, un saggio di legame peptide-MHC II scalata a 384 pozzetti è descritto. Questo formato conveniente, deve risultare utile nel campo della proteina deimmunization e progettazione di vaccini e sviluppo.

Un saggio di legame High Throughput MHC II per l&#39;analisi quantitativa dei peptidi epitopi

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

a-high-throughput-mhc-ii-binding-assay-for-quantitative-analysis

Research

JoVE Journal

Biology

14.8K Views.  Dartmouth College.  Saggi biochimici con molecole ricombinanti umani MHC II grado di fornire rapide, approfondimenti quantitativi nella identificazione degli epitopi immunogenico, la cancellazione o il design. Qui, un saggio di legame peptide-MHC II scalata a 384 pozzetti è descritto. Questo formato conveniente, deve risultare utile nel campo della proteina deimmunization e progettazione di vaccini e sviluppo. 

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

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

CD4 + antigene specifico visualizzando cellule T con MHC di classe II tetrameri

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

Ricerca

Biologia

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenesi e Analisi funzionale dei canali ionici Heterologously Espresso in cellule di mammifero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

High-throughput per l&#39;analisi Saccarificazione materiali lignocellulosici

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

A Quantitative Analysis Fitness Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

High-throughput screening funzionale utilizzando una casalinga Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

L&#39;analisi citofluorimetrica di bimolecular Complementation Fluorescenza: A High Throughput Metodo quantitativo per studiare l&#39;interazione proteina-proteina

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Analisi qPCRTag - A High Throughput, Real Time PCR Assay per Sc2.0 genotipizzazione

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

High Throughput<em&gt; Danio Rerio</em&gt; Energia spese Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...