1Department of Virology II, National Institute of Infectious Diseases, 2New Product Design Department, Fujirebio Inc.
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Arita, M., Masujima, S., Wakita, T., Shimizu, H. Particle Agglutination Method for Poliovirus Identification. J. Vis. Exp. (50), e2824, doi:10.3791/2824 (2011).
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).
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
3. Reconstitution of sensitized gelatin particle
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
|Well #1||Well #2||Well #3||Well #4||Well #5||Well #6|
|Well #1||Well #2||Well #3||Well #4||Well #5||Well #6|
|virus sample||virus sample||virus sample||virus sample||virus sample||GM|
5. Interpretation of the results
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).
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 μL, 8.2 x 107 CCID50 / 12 μL, and 3.5 x 107 CCID50 / 12 μL, respectively) were used for the identification by PA assay. No virus and no antibodies control showed precipitation of gelatin particles (non-agglutination, Negative control).
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 μL, 1.7 x 108 CCID50 / 25 μL, and 7.3 x 107 CCID50 / 25 μL, respectively) were diluted 1/20 to 1/320, and then 25 μL of diluted samples were examined by PA assay without anti-PV antibodies.
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) 7, 8, and successfully observed specific agglutination of the sensitized gelatin particles with PV isolates. With the high specificity and uniform affinity of the sensitized particles, the critical factors of PV identification in the PA assay are 1) virus titer (for valid detection of PV by PA assay) and 2) interpretation of agglutination (for correct identification of PV).
Generally, virus titer of PV isolates in the cell culture system is in a range of 106 to 8 CCID50 / 50 μL. The detection limits of PA assay (forming complete agglutination) for type 1, 2, and 3 PV were 1.5x106 CCID50, 5.3x105 CCID50, and 9.1x105 CCID50, respectively 2. The PA assay correctly identified most of the PV isolates even in the presence of other non-PV enteroviruses. However, for some samples contained a mixture of different serotypes of PV, minor serotype failed to be detected. Clear agglutination should be formed with sufficient virus titer above the detection limits. In case the agglutination in No antibodies sample was unclear or partial, the isolates should be re-amplified in RD cells to increase the titer 9.
Partial agglutination was observed when the virus titer was low and just below the detection limits. Clear difference of agglutination from negative control could be judged as positive in PA assay. However, quite subtle difference of the agglutination judged as positive could mislead the interpretation of results. Therefore, the results of Positive (partial agglutination) should be used as supportive information for possibly caused by minor serotypes of the isolates. However, this information would be helpful when following analysis (genomic sequencing of PV isolates or real-time RT-PCR) suggested a possibility that the sample contained a mixture of different serotypes of PV, or only partial agglutination was observed for No antibodies sample where the low virus titer would be the major cause of the unclear results.
The advantage of PA assay for PV identification is in the simplicity of the procedure and result interpretation. This would allow easy introduction of the assay to every laboratories conducting PV isolation with the cell culture system, without providing special equipment, materials, and trainings. Utilization of type-specific monoclonal antibodies might allow the development of novel method for the differentiation of PVs (wild type or vaccine-like strains) that would provide important information of the property of the PV isolates along with genetic differentiation by RT-PCR and genomic sequencing 10, 11.
Souji Masujima is an employee of New Product Design Department, Fujirebio Inc.
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.
|Sensitized-gelatin particles||Fujirebio Inc.||Test kit|
|Reconstitution buffer||Fujirebio Inc.||Test kit|
|Microtitration plate||Fujirebio Inc.||Test kit|