Development of a particle agglutination method with soluble virus receptor for identification of poliovirus

Abstract

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples of patients with acute flaccid paralysis (AFP). In this study, we developed a particle agglutination (PA) method with a soluble human PV receptor (hPVR) in the form of an immunoadhesin (PVR-IgG2a) for the simple and rapid identification of PV. Sensitized gelatin particles with PVR-IgG2a showed specific agglutination with the culture fluid of PV-infected cells within 2 h of reaction in a one-step procedure. Detection limits for type 1, 2, and 3 PV(Sabin) strains were 1.5 x 10(6) 50% cell culture infectious doses (CCID(50)), 5.3 x 10(5) CCID(50), and 9.1 x 10(5) CCID(50), respectively. Wild-type PVs and PV isolates from acute flaccid paralysis cases examined were identified correctly with this PA method, except for some samples with a mixture of different serotypes of PVs, where a minor population of PV failed to be detected. These results suggest that this PA method is useful for the simple and rapid identification of PV, although the sensitivity was not high enough to detect a minor population of PV (<1/10 of the major population) among mixed PVs.

FIG. 1.

PA method for the identification of PV. (A) Schematic view of a sensitized gelatin particle with a soluble PVR (PVR-IgG2a). (B) Procedure of the PA method for the identification of PV and the appearance of agglutination by PV. The order of sample addition to the reaction plate is as follows: 1, anti-PV antibodies; 2, PV solution; and 3, sensitized gelatin particle solution. r.t., room temperature.

FIG. 2.

Characterization of PA method and identification of PV strains. (A) Specificity of sensitized gelatin particles for PV. The agglutination activity of gelatin particles was examined with nonpoliovirus enteroviruses (echovirus 11 and CVB3) and nontreated gelatin particles. Wells that showed agglutination are shown in a box. (B) Sensitivity of the PA method for PV(Sabin) strains. Virus solutions of PV1(Sabin), PV2(Sabin), and PV3(Sabin) (virus titers of 9.5 × 10⁶ CCID₅₀/μl [1.1 × 10⁸ CCID₅₀ in 12 μl], 6.9 × 10⁶ CCID₅₀/μl [8.2 × 10⁷ CCID₅₀ in 12 μl], and 2.9 × 10⁶ CCID₅₀/μl [3.5 × 10⁷ CCID₅₀ in 12 μl], respectively) were diluted 1/5 to 1/640 and then examined by the PA method. The detection limit of the PA method for each sample observed on the reaction plate is shown with a box. (C) Identification of mixed PV(Sabin) strains by the PA method. Virus solutions of PV1(Sabin), PV2(Sabin), and PV3(Sabin) (virus titers of 9.5 × 10⁶ CCID₅₀/μl [1.1 × 10⁸ CCID₅₀ in 12 μl], 6.9 × 10⁶ CCID₅₀/μl [8.2 × 10⁷ CCID₅₀ in 12 μl], and 2.9 × 10⁶ CCID₅₀/μl [3.5 × 10⁷ CCID₅₀ in 12 μl], respectively) were mixed with a 1/10 volume (1.2 μl) of each PV(Sabin) strain and then subjected to the PA method for identification. (D) Identification of wild-type PV strains by the PA method. Virus titers of wild-type strains examined [PV1(Brunhilde), PV1(Mahoney), PV2(MEF-1), PV3(Leon), PV3(Saukett), and PV3(Suwa-3)] were 3.2 × 10⁶ CCID₅₀/μl, 1.8 × 10⁶ CCID₅₀/μl, 1.0 × 10⁷ CCID₅₀/μl, 1.0 × 10⁶ CCID₅₀/μl, 1.0 × 10⁶ CCID₅₀/μl, and 5.6 × 10⁵ CCID₅₀/μl, respectively.