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 antibody (type 1, 2, or 3). Add this reconstituted antibody solutions (0.5 mL each) to 4.5 mL of MM in separate labeled tubes. Note: Some lots of anti-PV antibody are provided as reconstituted form (0.5 mL per vial). For such lot of anti-PV antibody, addition of water for reconstitution is not necessary.</li>
<li>Mix equal volume of reconstituted anti-PV antibody. For anti-PV1 +2, 2+3, or 3+1: Mix 1.8 mL of each reconstituted antibody (1.8 mL + 1.8 mL = 3.6 mL for each mixture). For anti-PV1 +2+3: Mix 1.2 mL of each reconstituted antibody (1.2 mL + 1.2 mL + 1.2 mL = 3.6 mL). Note: PV samples could contain a mixture of different serotypes of PV. To identify single serotype of PV in samples with mixture of PVs, pools of anti-PV antibodies (i.e. 1+2, 2+3, 3+1, 1+2+3) is necessary.</li>
<li>Add 1/5 volume of FCS (0.72 mL) to each anti-PV (1+2, 2+3, 3+1, or 1+2+3) antibody (3.6 mL each) (final 18.2% FCS). Note: Addition of FCS is for reduction of non-specific agglutination of gelatin particle caused by antibodies.</li>
<li>Keep the antibody pools at -20 &deg;C until use in PA assay.</li>
</ol>
3.	Reconstitution of sensitized gelatin particle
<ol>
<li>Add 1.5 mL of reconstitution buffer included in the kit to one vial of sensitized gelatin particle (provided as freeze-dried powder).</li>
<li>Mix well by gentle tapping</li>
<li>Keep the reconstituted sensitized particle at 4 &deg;C. Reconstituted gelatin particle could be active at least 2 weeks kept at 4 &deg;C, but should be prepared just before the assay.</li>
</ol>
Important note: 
Do not mix different lot of sensitized gelatin particles, because the non-agglutination image of sensitized gelatin particles could be different by the lot of the particles. For each experiment, a single lot of sensitized gelatin particle should be used.

4.	PA assay for PV identification
<ol>
<li>Prepare a micro-titration plate. For identification of one virus sample, we need 5 wells + at least one negative control (sensitized particle without virus and antibody, well # 6 in below. See No virus and antibody control in Example). Total number of wells is 5 x samples + at least one control in the assay. Important note: 
Please take at least one No virus and antibody control in the assay. One vial of sensitized particle is approximately for identification of 10 virus samples.</li>
<li>Add 12 &mu;L/well of anti-PV antibody or GM to the micro-titration plate. The arrangement of the plate is as follows (See also Example below). In samples without antibody (well #1 and 6), add 12 &mu;L of GM instead of anti-PV antibody. 
Important note: 
If many PV samples should be tested in this assay, multichannel pipette or dispenser would be helpful for this step.
<table border="1">
	<tbody>
 	<tr>
 	<td>Well #1</td>
 <td>Well #2</td>
 <td>Well #3</td>
 <td>Well #4</td>
 <td>Well #5</td>
 <td>Well #6</td>
 </tr>
 <tr>
 	<td>GM</td>
 <td>anti-P1+2+3</td>
 <td>anti-P1+2</td>
 <td>anti-P2+3</td>
 <td>ani-P1+3</td>
 <td>GM</td>
 </tr>

 </tbody>
</table>
</li>
<li>Add 12 &mu;L/well of virus sample. In one sample without virus (well #6), add 12 &mu;L of GM instead of virus solution. Note: Virus sample is culture supernatant of infected cells, usually L20B cells or RD cells. 
Important note: 
If many PV samples should be tested in this assay, multichannel pipette would be helpful for this step.
<table border="1">
	<tbody>
 	<tr>
 	<td>Well #1</td>
 <td>Well #2</td>
 <td>Well #3</td>
 <td>Well #4</td>
 <td>Well #5</td>
 <td>Well #6</td>
 </tr>
 <tr>
 	<td>virus sample</td>
 <td>virus sample</td>
 <td>virus sample</td>
 <td>virus sample</td>
 <td>virus sample</td>
 <td>GM</td>
 </tr>

 </tbody>
</table> 
</li> 
<li>Take reconstituted sensitized particle solution from 4 &deg;C to room temperature, and gently tap to completely suspend the particles. Important note: After complete suspension of the particles by gentle tapping, the particles should be added immediately to the plates before the suspended particles precipitate and form non-homogeneous solution.</li>
<li>Add 25 &mu;L/well of sensitized particle to all the wells. Important note: After addition of sensitized particle, the plate should immediately be subjected to plate mixing to start uniform reaction of the samples. Therefore, addition of sensitized particle should be performed by using dispenser to minimize a time lag among the samples in the plate.</li> 
<li>Put the micro-titration plate onto a plate mixer and mix the plate for 30 sec.</li>
<li>Put the micro-titration plate on white paper (for convenience of visual observation) on a table at room temperature (15&tilde;30 &deg;C) for 2 h. Important note: 
Do not move the plate during and after the reaction for observation of the results and taking photos. The formed agglutination is not stable and easily disturbed by moving the plates.</li>
<li>Take photos of the plate. 
Important note: 
Partial view of the photos could not give sufficient data for later examination and review of the results for interpretation. Several photos should be taken to cover all the wells of the plates focusing on the agglutination image of the wells.</li>
</ol>
5. Interpretation of the results
<ol>
<li>Identification of positive and negative wells. Compare the agglutination in the samples with that of No virus and antibody control (precipitation of gelatin particles, non-agglutination) (Figure 1 and 2). Images of agglutination that are clearly distinguished from No virus and no antibody control (non-agglutination, negative control) should be judged as positive, and images of agglutination that are similar to the negative control should be judged as negative. Samples that show clear but only partial agglutination could be judged as positive, but record as 'Positive (partial agglutination)'.</li> 
<li>Identification of PV. Interpret the results based on positive and negative wells with anti-PV antibodies to identify PV serotypes. Principle of the interpretation of the result is similar to that of neutralization test in cell culture system (e.g. positive for anti-P1+2 antibody indicates that the sample contains type 3 PV).</li>
</ol>
6. Representative Results
Representative result of PA assay PV identification is shown in Figure 1. All the PV strains formed agglutination of sensitized gelatin particles (No antibodies samples). In the presence of corresponding anti-PV antibodies, precipitation of gelatin particles (non-agglutination) similar to No virus and no antibodies control (negative control) was observed (e.g., PV1(Sabin) in the presence of anti-P1+2 or anti-P3+1 antibodies). 
<img src="/files/ftp_upload/2824/2824fig1.jpg" alt="Figure 1" /> 
Figure 1. Identification of PV by PA assay. Virus solutions of PV1(Sabin), PV2(Sabin), and PV3(Sabin) (virus titers of 1.2 x 108 CCID50 / 12 &mu;L, 8.2 x 107 CCID50 / 12 &mu;L, and 3.5 x 107 CCID50 / 12 &mu;L, respectively) were used for the identification by PA assay. No virus and no antibodies control showed precipitation of gelatin particles (non-agglutination, Negative control).
<img src="/files/ftp_upload/2824/2824fig2.jpg" alt="Figure 2" /> 
Figure 2. Interpretation of Positive and Negative wells. Virus solutions of PV1(Sabin), PV2(Sabin), and PV3(Sabin) (virus titers of 2.4 x 108 CCID50 / 25 &mu;L, 1.7 x 108 CCID50 / 25 &mu;L, and 7.3 x 107 CCID50 / 25 &mu;L, respectively) were diluted 1/20 to 1/320, and then 25 &mu;L of diluted samples were examined by PA assay without anti-PV antibodies.

<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>

Souji Masujima is an employee of New Product Design Department, Fujirebio Inc.

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 ...

particle-agglutination-method-for-poliovirus-identification

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Immunology and Infection

15.2K Views.  National Institute of Infectious Diseases. 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.