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.
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Method Article
Particle Agglutination Method for Poliovirus Identification
In This Article
Summary
Abstract
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).
Protocol
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
- 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 ....
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Discussion
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˜-8 M) <.......
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Disclosures
Souji Masujima is an employee of New Product Design Department, Fujirebio Inc.
Acknowledgements
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.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Sensitized-gelatin particles | Fujirebio Inc. | Test kit | |
| Reconstitution buffer | Fujirebio Inc. | Test kit | |
| Microtitration plate | Fujirebio Inc. | Test kit | |
| Anti-PV antibody | RIVM |
References
- World Health Organization. Polio Laboratory Manual (4th edition) WHO/IVB/04.10. , 4th Edition, World Health Organization. (2004).
- Arita, M., Masujima, S., Wakita, T., Shimizu, H. Development of a particle agglutination method with soluble virus receptor for identification of poliovirus. ....
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