Questo studio è stato sostenuto dal National Institutes of Health (NIH) degli Stati Uniti (RO1 AI68002).

1. Ricombinante SARS-CoV RBD proteine ​​preparazione <ol><li> Preparare fosfato di calcio dei reagenti di trasfezione <ol><li> 2X preparazione tampone HBS: Mescolare 16 g di NaCl, 0,4 g di Na 2 HPO 4 * 7H 2 O e 13,0 g di HEPES. Regolare il pH a 7,00 e portare il volume totale di 1000 ml di acqua distillata. Dopo il filtraggio la soluzione per la sterilizzazione, aliquota e conservarlo a -20 ° C. Suggerimento: Ogni variazione del valore di pH potrebbe influenzare i risultati di trasfezione. Quindi, si consiglia di testare diversi valori di pH intorno a 7,00 (per esempio, 6,99, 7,00 o 7,01) e trovare la migliore per la trasfezione con il metodo introdotto di seguito. </li><li> 2,5 M CaCl 2 Preparazione: Aggiungere 73,5 g di CaCl 2 * 2H 2 O in acqua distillata in un volume finale di 200 mL. Filtrare la soluzione e conservare a -20 ° C. </li></ol></li><li> Trasfezione plasmide ricombinante e purificazione di proteine <ol><li> Dividere le celle 293T al 50-70% confluenza 24 ore prima di trasfezione. Crescere le cellule di T-175 palloni tessuto cm 2 cultura in 40 ml di DMEM contenente 10% di siero (HI) FBS e 1% di penicillina / streptomicina (P / S) a 37 ° C in 5% di CO 2. </li><li> Tutti i reagenti di trasfezione deve essere portato a temperatura ambiente prima di trasfezione. Questi reagenti sono HBS 2X, 2.5M CaCl 2, DIH 2 O, e rRBD plasmide 6. Suggerimento: Il plasmide ricombinante finale costrutto usato per l&#39;espressione delle proteine ​​dovrebbe contenere un peptide segnale per garantire la secrezione di proteine ​​ricombinanti espresse al supernatante della coltura. Un 6x suoi tag possono essere aggiunti al C-terminale della proteina espressa per la purificazione facile. </li><li> Preparare un 50 ml (A) e uno da 15 ml (B) BD tubo Falcon, e aggiungere i reagenti per i tubi, come indicato nella tabella 1. Aggiungi 2X tampone HBS ad A. Tubo in tubo B, aggiungere 2,5 M CaCl 2 e rRBD plasmide, e portare il volume al requisito in DIH 2 O. <table border="1"><tbody><tr><td colspan="4"> A. Un T-175 cm 2 cultura fiasco tessuti (4000 microlitri / cilindro): prepararsi per l&#39;espressione rRBD </td></tr><tr><td colspan="2"> Un tubo </td><td colspan="2"> Tubo B </td></tr><tr><td> HBS 2X </td><td> 2000 microlitri </td><td> 2,5 M CaCl 2 </td><td> 200 l </td></tr><tr><td></td><td></td><td> rRBD DNA plasmidico </td><td> 40 mcg </td></tr><tr><td></td><td></td><td> DIH 2 O a </td><td> 2000 microlitri </td></tr><tr><td colspan="4"> B. One 100 mm scatola di Petri (1000 ml / piatto): prepararsi per la produzione di pseudovirus SARS </td></tr><tr><td colspan="2"> Un tubo </td><td colspan="2"> Tubo B </td></tr><tr><td> HBS 2X </td><td> 500 microlitri </td><td> 2,5 M CaCl 2 </td><td> 50 microlitri </td></tr><tr><td></td><td></td><td> DNA plasmidico SARS-CoV S </td><td> 5 mcg </td></tr><tr><td></td><td></td><td> HIV-1 plasmide (pNL4-3.luc.RE) </td><td> 5 mcg </td></tr><tr><td></td><td></td><td> DIH 2 O a </td><td> 500 microlitri </td></tr></tbody></table> Tabella 1. Miscela di preparazione Transfection e volumi I volumi elencati nella Tabella 1 sono per una unità di trasfezione. Se più fiaschi o piatti vengono utilizzati per la transfezione, regolare i volumi di conseguenza. </li><li> Aggiungere il DNA di calcio soluzione in B tubo nel tubo A in maniera goccia a goccia, pur mantenendo costante e dolce mescolando in un vortice. Lasciate il composto a temperatura ambiente per 20-30 min. La chiave per il successo di trasfezione fosfato di calcio dipende dalle dimensioni e la forma del precipitato formato. Così, la miscela deve essere costantemente in agitazione e lentamente per soddisfare questo requisito e, di conseguenza, migliorare l&#39;efficacia di trasfezione. </li><li> Aggiungete il composto in maniera goccia a goccia e anche in cellule 293T (4000 microlitri / fiasco). Cellule cultura in incubatore a 37 ° C in 5% di CO 2. </li><li> Sostituire il terreno di coltura fresca senza siero OPTI-MEM ho ridotto-siero Media (50 mL / beuta) 8-10 ore dopo la trasfezione. Continuare a celle di coltura per due giorni nelle stesse condizioni. </li><li> Raccogliere il supernatante contenente proteine ​​espresse rRBD 72 h post-trasfezione. Centrifugare a 6000 rpm per 15 minuti per rimuovere i detriti cellulari. Aggiungi cocktail inibitore della proteasi per il supernatante raccolto e conservare a 4 ° C durante la notte. </li><li> Il giorno dopo, purificare rRBD proteina ricombinante dal supernatante della coltura utilizzando le istruzioni Ni-NTA produttore Superflow successivo. </li><li> Concentrato di proteine ​​purificate utilizzando Amicon Ultra tubi concentrazione -15. Dopo la concentrazione di proteine,aggiungere PBS ai tubi concentrazione e centrifugare di nuovo per rimuovere imidazolo in tampone di eluizione. Calcolare la concentrazione di proteine ​​e conservare le proteine ​​purificate a -80 ° C fino al momento dell&#39;uso. </li></ol></li></ol> 2. Immunizzazione del mouse e Prelievo dei campioni <ol><li> Preriscaldare Sigma sistema adiuvante (SAS) a 40-45 ° C secondo le istruzioni del produttore. Aggiungere 1 ml di PBS per flacone e mescolare accuratamente. </li><li> Preparare proteina-adiuvante emulsione secondo il protocollo nella tabella 2. Pipetta calcolato proteina rRBD in un tubo di 1,5 ml. Aggiungere uguale volume di SAS adiuvante per il tubo e agitare vigorosamente per 2-3 minuti per formare emulsione. Suggerimento: I volumi elencati nella tabella 2 sono per un mouse. Regolare i volumi secondo i numeri del mouse effettivamente utilizzato. Per ogni gruppo, si mescolano le proteine ​​e adiuvante necessari per ogni vaccino. Sempre preparare un campione in più per la vaccinazione per garantire l&#39;accuratezza. <table border="1"><tbody><tr><td> Gruppo </td><td> 1 ° vaccino (200 l / mouse) </td><td> 2 ° vaccino (200 l / mouse) </td><td> 3 ° vaccino (200 l / mouse) </td></tr><tr><td> rRBD proteina </td><td> 20 mg di proteine ​​in PBS (100 mL) + 100 ul SAS </td><td> 10 mg di proteine ​​in PBS (100 mL) + 100 ul SAS </td><td> 10 mg di proteine ​​in PBS (100 mL) + 100 ul SAS </td></tr><tr><td> PBS controllo </td><td> 100 ul PBS + 100 ul SAS </td><td> 100 ul PBS + 100 ul SAS </td><td> 100 ul PBS + 100 ul SAS </td></tr></tbody></table> Tabella 2. Mouse immunizzazione protocollo </li><li> Per via sottocutanea in prima serata immunizzare femminile topi BALB / c (4-6 settimane, 5 topi / gruppo), e aumentare due volte con rRBD e SAS, come indicato nella tabella 2. Usa PBS gruppo come il controllo. Il sito per iniezioni sottocutanee di solito scelto è la pelle flaccida tra le scapole. In alternativa, l&#39;addome ventrale è comunemente usato, perché è più facile da iniettare, e può osservare eventuali perdite dai siti di iniezione. Quando dosi ripetute di vaccini vengono utilizzati, è facile selezionare i siti di iniezione diversi, evitando potenziali reazioni cutanee locali. </li><li> Bleed topi via retro-orbitale con anestetico prima vaccinazione e 10 giorni dopo ogni vaccinazione, e il calore siero a 56 ° C per 30 minuti per inattivare il complemento. Conservare il mouse siero a -20 ° C fino al momento dell&#39;uso. </li></ol> 3. Neutralizzazione di rilevamento utilizzando neutralizzazione saggio Pseudovirus <ol><li> Preparare pseudovirus SARS <ol><li> Dividere le celle 293T in 100 piatti della cultura millimetro di tessuto di Petri (2x10 6 cellule / piatto) 16 h prima di trasfezione, le cellule e far crescere come sopra. </li><li> Preparare i reagenti di trasfezione, come indicato nella tabella 1. Proteine ​​co-trasfezione una codifica plasmide SARS-CoV S e una codifica plasmide Env-difettoso, luciferasi che esprimono genoma dell&#39;HIV-1 (pNL4-3.luc.RE) con fosfato di calcio dei reagenti di trasfezione. </li><li> Sostituire media con 10 ml fresca DMEM contenente 10% FBS e 1% P / S 8-10 ore dopo la trasfezione. Raccogliere il supernatante contenente pseudovirus SARS 72 h post-trasfezione. </li><li> Pseudovirus filtrare attraverso un filtro da 0,45 micron. Aliquota e conservare a -80 ° C fino al momento dell&#39;uso. </li></ol></li><li> SARS pseudovirus test di neutralizzazione <ol><li> Dividere le celle 293T esprimono SARS-CoV recettore ACE2 (ACE2/293T) a 10 4 cells/100 microlitri / pozzetto in piastre da 96 pozzetti tessuto cultura 16 h prima dell&#39;infezione. </li><li> Diluire pseudovirus SARS da 2 volte a rilevare il titolo del virus nelle cellule ACE2/293T. </li><li> Serialmente diluire i sieri del mouse in lastre di tessuto da 96 pozzetti cultura, e aggiungere uguale volume di titolato pseudovirus SARS. Preincubare le piastre a 37 ° C per 1 h. </li><li> Dopo l&#39;incubazione, aggiungere 100 microlitri della Sera-pseudovirus miscela di cellule ACE2/293T, e continuano a crescere le cellule a 37 ° C in 5% di CO 2. Aggiungi fresca DMEM 24 ore più tardi. </li><li> Rimuovere completamente sovranatanti dalle piastre 72 ore dopo l&#39;infezione. Aggiungi 1X luciferasi cultura reagente di lisi cellulare (60 microlitri / pozzetto), e promuovere la lisi cellulare con costante agitazione di piastre per 1-2 ore a temperatura ambiente. </li><li> Trasferimento lisati cellulari (50 ml / pozzetto) in piastre luminometro (Microfluor piastre a 96 pozzetti). Aggiungere substrato luciferasi (50 ml / pozzetto) inclusi nel sistema di analisi luciferasi, e di rilevare l&#39;attività relativa luciferasi in Ultra 384 luminometro. </li><li> Calcolare titolo pseudovirus SARS neutralizzazione e presente come il 50% titolo di anticorpi neutralizzanti (NT 50) 6. </li></ol></li></ol>

<ol>
	<li>Pitcovski, J., Gutter, B., Gallili, G., Goldway, M., Perelman, B., Gross, G., Krispel, S., Barbakov, M., Michael, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+large-scale+use+of+recombinant+VP2+vaccine+for+the+prevention+of+infectious+bursal+disease+of+chickens.">Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens.</a> Vaccine. 21, 4736-4743 (2003).</li><li>Tait, A., Hall, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theileria+annulata:+control+measures,+diagnosis+and+the+potential+use+of+subunit+vaccines.">Theileria annulata: control measures, diagnosis and the potential use of subunit vaccines.</a> Rev. Sci. Tech. 9, 387-403 (1990).</li><li>Qi, Z., Zhou, L., Zhang, Q., Ren, L., Dai, R., Wu, B., Wang, T., Zhu, Z., Yang, Y., Cui, B., Wang, Z., Wang, H., Qiu, Y., Guo, Z., Yang, R., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mouse,+guinea+pig+and+rabbit+models+for+evaluation+of+plague+subunit+vaccine+F1+rV270.">Comparison of mouse, guinea pig and rabbit models for evaluation of plague subunit vaccine F1+rV270.</a> Vaccine. 28, 1655-1660 (2010).</li><li>Rosser, M. P., Xia, W., Hartsell, S., McCaman, M., Zhu, Y., Wang, S., Harvey, S., Bringmann, P., Cobb, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transfection+of+CHO-K1-S+using+serum-free+medium+in+suspension:+a+rapid+mammalian+protein+expression+system.">Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system.</a> Protein Expr. Purif. 40, 237-243 (2005).</li><li>Du, L., Zhao, G., Li, L., He, Y., Zhou, Y., Zheng, B. J., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenicity+and+immunogenicity+of+SARS-CoV+S+protein+receptor-binding+domain+stably+expressed+in+CHO+cells.">Antigenicity and immunogenicity of SARS-CoV S protein receptor-binding domain stably expressed in CHO cells.</a> Biochem. Biophys. Res. Commun. 384, 486-490 (2009).</li><li>Du, L., Zhao, G., Chan, C. C., Sun, S., Chen, M., Liu, Z., Guo, H., He, Y., Zhou, Y., Zheng, B. J., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+receptor-binding+domain+of+SARS-CoV+spike+protein+expressed+in+mammalian,+insect+and+E.+coli+cells+elicits+potent+neutralizing+antibody+and+protective+immunity.">Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity.</a> Virology. 393, 144-150 (2009).</li><li>Ambrosino, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+270+to+510+of+the+severe+acute+respiratory+syndrome+coronavirus+spike+protein+are+required+for+interaction+with+receptor.">Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor.</a> J. Virol. 78, 4552-4560 (2004).</li><li>Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., Choe, H., Farzan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-converting+enzyme+2+is+a+functional+receptor+for+the+SARS+coronavirus.">Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.</a> Nature. 426, 450-454 (2003).</li><li>He, Y., Zhou, Y., Liu, S., Kou, Z., Li, W., Farzan, M., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-binding+domain+of+SARS-CoV+spike+protein+induces+highly+potent+neutralizing+antibodies:+implication+for+developing+subunit+vaccine.">Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine.</a> Biochem. Biophys. Res. Commun. 324, 773-781 (2004).</li><li>Batard, P., Jordan, M., Wurm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+high+copy+number+plasmid+into+mammalian+cells+by+calcium+phosphate+transfection.">Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection.</a> Gene. 270, 61-68 (2001).</li><li>Pramfalk, C., Lanner, J., Andersson, M., Danielsson, E., Kaiser, C., Renstrom, I. M., Warolen, M., James, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+receptor+activation+and+down-regulation+by+cationic+lipid+transfection+reagents.">Insulin receptor activation and down-regulation by cationic lipid transfection reagents.</a> BMC. Cell Biol. 5, 7-7 (2004).</li><li>Rosser, M. P., Xia, W., Hartsell, S., McCaman, M., Zhu, Y., Wang, S., Harvey, S., Bringmann, P., Cobb, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transfection+of+CHO-K1-S+using+serum-free+medium+in+suspension:+a+rapid+mammalian+protein+expression+system.">Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system.</a> Protein Expr. Purif. 40, 237-243 (2005).</li><li>Jiang, M., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Ca2+-phosphate+transfection+efficiency+in+low-density+neuronal+cultures.">High Ca2+-phosphate transfection efficiency in low-density neuronal cultures.</a> Nat. Protoc. 1, 695-700 (2006).</li><li>Jiang, M., Deng, L., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Ca(2+)-phosphate+transfection+efficiency+enables+single+neuron+gene+analysis.">High Ca(2+)-phosphate transfection efficiency enables single neuron gene analysis.</a> Gene Ther. 11, 1303-1311 (2004).</li><li>Han, D. P., Kim, H. G., Kim, Y. B., Poon, L. L., Cho, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+safe+neutralization+assay+for+SARS-CoV+and+characterization+of+S-glycoprotein.">Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein.</a> Virology. 326, 140-149 (2004).</li><li>Toledo, J. R., Prieto, Y., Oramas, N., Sanchez, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-based+transfection+method+as+a+simple+and+effective+way+to+produce+recombinant+lentiviral+vectors.">Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors.</a> Appl. Biochem. Biotechnol. 157, 538-544 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Densità delle cellule è un fattore importante per l&#39;efficacia di fosfato di calcio a base di trasfezione. Nella nostra esperienza, meno del 70% confluenza delle cellule porta i migliori risultati. Pertanto, al fine di migliorare l&#39;efficienza di trasfezione, si raccomanda che la densità delle cellule mantenuto a circa 50-70% di confluenza. Il fosfato di calcio metodo di trasfezione è generalmente ritenuto meno efficiente rispetto ai metodi di transfezione altri, come la transfezione lipidi 16. Tuttavia, in questo protocollo, abbiamo usato una versione modificata del metodo di fosfato di calcio trasfezione cui costante e lenta miscelazione della soluzione di trasfezione in un vortice assicura la formazione di un precipitato con la dimensione appropriata e la forma, migliorando così l&#39;efficienza di trasfezione.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4*7H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2429</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2*2H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C5080</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12430</td>
<td>&nbsp;</td>
<tr>
<td>HI FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10438</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>OPTI-MEM I Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836170001</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA Superflow</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>30450</td>
<td>&nbsp;</td>
<tr>
<td>Sigma adjuvant system</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6322</td>
<td>&nbsp;</td>
<tr>
<td>Luciferase assay system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1501</td>
<td>&nbsp;</td>
<tr>
<td>Luciferase cell culture lysis reagent (5X)</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1531</td>
<td>&nbsp;</td>
<tr>
<td>Amicon Ultra - 15</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC901024</td>
<td>&nbsp;</td>
<tr>
<td>Microfluor 96-well plates</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>7905</td>
<td>&nbsp;</td>
<tr>
<td>Ultra 384 luminometer</td>
<td>&nbsp;</td>
<td>Tecan Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

protocol for recombinant rbd-based sars vaccines: protein preparation, animal vaccination and neutralization detection

Sulla base del loro profilo di sicurezza e la capacità di indurre potenti risposte immunitarie contro le infezioni, vaccini a subunità sono stati usati come candidati per una vasta gamma di agenti patogeni 1-3. Dato che il sistema delle cellule di mammifero è in grado di modificazioni post-traduzionali, in modo da formare le proteine ​​accuratamente piegati e glicosilata, proteine ​​ricombinanti espresse in cellule di mammifero hanno dimostrato il potenziale maggiore per mantenere alta l&#39;antigenicità e immunogenicità 4-6. Sebbene non nuovi casi di SARS sono stati segnalati dal 2004, future epidemie sono una minaccia costante, quindi, lo sviluppo di vaccini contro la SARS-CoV è un passo prudente preventiva e deve essere effettuata. L&#39;RBD di proteine ​​SARS-CoV S svolge un ruolo importante nel legame recettore e induzione di specifici anticorpi neutralizzanti contro l&#39;infezione da virus 7-9. Pertanto, in questo protocollo, si descrivono nuovi metodi per lo sviluppo di un vaccino basato RBD subunità contro la SARS. In breve, la proteina ricombinante RBD (rRBD) è stato espresso in supernatante della coltura di cellule di mammifero 293T per ottenere una proteina correttamente ripiegata con conformazione corretta e l&#39;immunogenicità di alta 6. La transfezione della codifica RBD plasmide ricombinante per le cellule è stata poi effettuata utilizzando un metodo di fosfato di calcio trasfezione 6,10 con alcune modifiche. Rispetto al metodo di trasfezione lipidi 11,12, questo metodo modificato fosfato di calcio transfezione è più conveniente, più facile da gestire, e ha il potenziale per raggiungere elevata efficacia una volta un complesso di transfezione con dimensioni adatte e la forma è formata 13,14. Infine, un saggio di neutralizzazione pseudovirus SARS è stato introdotto nel protocollo e utilizzati per rilevare l&#39;attività neutralizzante di sieri di topi vaccinati con proteine ​​rRBD. Questo test è relativamente sicuro, non comporta alcuna infettive SARS-CoV, e può essere eseguito senza la necessità di un laboratorio di biosicurezza-3 15. Il protocollo qui descritto può essere utilizzato anche per la progettazione e lo studio vaccini a subunità ricombinanti contro altri virus con classe I proteine ​​di fusione, per esempio, l&#39;HIV, il virus respiratorio sinciziale (RSV), virus Ebola, virus dell&#39;influenza, così come virus Nipah e Handra. Inoltre, i metodi per generare un pseudovirus e, successivamente, che istituisce un saggio di neutralizzazione pseudovirus può essere applicato a tutti questi virus.

Watch this Scientific Journal Video about Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection at JoVE.com

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Questo protocollo descrive una procedura generale per lo studio ricombinante del recettore dominio di legame (RBD) a base di vaccini a subunità contro la SARS. Esso include metodi per la trasfezione ed espressione di proteine ​​nelle cellule 293T RBD, l&#39;immunizzazione di topi con RBD e la rilevazione di attività di neutralizzazione del mouse utilizzando un siero stabilito pseudovirus SARS saggio di neutralizzazione.

Protocollo per ricombinante RBD-based Vaccines SARS: Preparazione di proteine, vaccinazione degli animali e la rivelazione di neutralizzazione

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

protocol-for-recombinant-rbd-based-sars-vaccines-protein-preparation

Research

JoVE Journal

Immunology and Infection

42.4K Views.  New York Blood Center.  中文 

Video: Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

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

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

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

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

Combinazione di nastro adesivo a base di campionamento e di fluorescenza In situ</emIbridazione> per il rilevamento rapido di Salmonella Su prodotti freschi

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

Ricerca

Immunologia e infezioni

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Imaging Whole-animale e di citometria a flusso tecniche di analisi di Antigen-specific CD8 + cellule T Risposte dopo nanoparticelle vaccinazione

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Intranasale somministrazione di ricombinante Influenza vaccini nei modelli chimerico topo per studiare immunità delle mucose

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

In Vivo Test per la determinazione della funzione citolitica di cellule T antigene-specifiche utilizzando un modello di vaccinazione

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Generazione di vettori ricombinanti Herpesvirus aviaria con CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Analisi degli analoghi dell'acido iofenostico nella piccola mangusta indiana (Herpestes Auropunctatus) Sera per l'uso come marcatore biologico di vaccinazione della rabbia orale

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

Un test di neutralizzazione rapido e multiplex Dual Reporter IgG e IgM SARS-CoV-2 per un sistema di analisi del flusso basato su perle multiplexate

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

Rilevazione dell'anticorpo sars-CoV-2 Receptor-Binding Domain utilizzando un bioreporter basato su HiBiT

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

Imaging dal vivo e quantificazione dell'infezione virale in topi transgenici K18 hACE2 utilizzando SARS-CoV-2 ricombinante che esprime reporter

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

Rilevamento di epitopi sensibili alla neutralizzazione in antigeni visualizzati su vaccini basati su particelle simili a virus (VLP) utilizzando un test di cattura

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...