Name: particle agglutination method for poliovirus identification
Uploaded: 2011-04-20T14:00:00
Description: Watch this Scientific Journal Video about Particle Agglutination Method for Poliovirus Identification at JoVE.com

We are grateful to Junko Wada for her excellent technical assistance. We are grateful to Prof. Akio Nomoto for kindly providing a baculovirus expression vector for PVR-IgG2a. This study was supported in part by Grants-in-Aid for the Promotion of Polio Eradication and Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labour and Welfare.

1.	Preparation of maintenance medium (MM) and growth medium (GM)
For preparation of MM and GM, please see a Polio laboratory manual of WHO 1. Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% fetal calf serum (FCS) and 10% FCS could be used as substitutes of MM and GM, respectively.
2.	Preparation of anti-PV antibodies
<ol>
<li>Reconstitute anti-PV antibody (RIVM PV typing antiserum) by adding water (0.5 mL per vial) to each vial of anti-PV ...

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Polio Laboratory Manual (4th edition) WHO/IVB/04.10. , (2004).</li><li>Arita, M., Masujima, S., Wakita, T., Shimizu, H. <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+particle+agglutination+method+with+soluble+virus+receptor+for+identification+of+poliovirus.">Development of a particle agglutination method with soluble virus receptor for identification of poliovirus.</a> J. Clin. Microbiol. 48, 2698-2702 (2010).</li><li>Bibb, J. A., Witherell, G., Bernhardt, G., Wimmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+cell+surface+binding+site.">Interaction of poliovirus with its cell surface binding site.</a> Virology. 201, 107-115 (1994).</li><li>Koike, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poliovirus+receptor+protein+is+produced+both+as+membrane-bound+and+secreted+forms.">The poliovirus receptor protein is produced both as membrane-bound and secreted forms.</a> Embo J. 9. , 3217-3224 (1990).</li><li>Mendelsohn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+a+human+poliovirus+receptor+gene+into+mouse+cells.">Transformation of a human poliovirus receptor gene into mouse cells.</a> Proc. Natl. Acad. Sci. U S A. 83, 7845-7849 (1986).</li><li>Mendelsohn, C. L., Wimmer, E., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+receptor+for+poliovirus:+molecular+cloning,+nucleotide+sequence,+and+expression+of+a+new+member+of+the+immunoglobulin+superfamily.">Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.</a> Cell. 56, 855-865 (1989).</li><li>Arita, M., Koike, S., Aoki, J., Horie, H., Nomoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+purified+receptor+and+conformational+alteration+in+the+virion.">Interaction of poliovirus with its purified receptor and conformational alteration in the virion.</a> J. Virol. 72, 3578-3586 (1998).</li><li>McDermott, B. M., Rux, A. H., Eisenberg, R. J., Cohen, G. H., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+binding+affinities+of+poliovirus+for+its+cellular+receptor.">Two distinct binding affinities of poliovirus for its cellular receptor.</a> J. Biol. Chem. 275, 23089-23096 (2000).</li><li>Pipkin, P. A., Wood, D. J., Racaniello, V. R., Minor, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+L+cells+expressing+the+human+poliovirus+receptor+for+the+specific+detection+of+polioviruses+in+vitro.">Characterisation of L cells expressing the human poliovirus receptor for the specific detection of polioviruses in vitro.</a> J. Virol. Methods. 41, 333-340 (1993).</li><li>Avoort, H. G. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+five+methods+for+intratypic+differentiation+of+polioviruses.">Comparative study of five methods for intratypic differentiation of polioviruses.</a> J. Clin. Microbiol. 33, 2562-2566 (1995).</li><li>Kilpatrick, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+group-,+serotype-,+and+vaccine+strain-specific+identification+of+poliovirus+isolates+by+real-time+reverse+transcription-PCR+using+degenerate+primers+and+probes+containing+deoxyinosine+residues.">Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.</a> J. Clin. Microbiol. 47, 1939-1941 (2009).</li></ol>

The unique point of this PA assay is the utilization of PVR molecule instead of anti-PV antibodies for the detection of PV 2. This allows a specific interaction of the sensitized gelatin particles with PV with a uniform affinity to all three serotypes of PV 3-6. We used an immunoadhesin form of PVR (PVR-IgG2a) for the sensitization of gelatin particles, because monomeric forms of PVR have only moderate affinities to the single binding site on the virion (dissociation constant of 10-7&tilde;-8 M) <...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 	<td>Sensitized-gelatin particles</td>
 <td>Fujirebio Inc.</td>
 <td>&nbsp;</td>
 <td>Test kit</td>
 
 </tr>
 <tr>
 	<td>Reconstitution buffer</td>
 <td>Fujirebio Inc.</td>
 <td>&nbsp;</td>
 <td>Test kit</td>
 
 </tr>
 <tr>
 	<td>Microtitration plate</td>
 <td>Fujirebio Inc.</td>
 <td>&nbsp;</td>
 <td>Test kit</td>
 
 </tr>
 <tr>
 	<td>Anti-PV antibody</td>
 <td>RIVM</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 
 </tr>
 
 </tbody>
</table>

particle agglutination method for poliovirus identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

Watch this Scientific Journal Video about Particle Agglutination Method for Poliovirus Identification at JoVE.com

Particle Agglutination Method for Poliovirus Identification

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

Research

JoVE Journal

Immunology and Infection

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...