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A microtitre plate method for isolation and typing of poliovirus using a blue-cell ELISA
1Enteric, Respiratory and Neurological Virus Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, NW9 5HT, London, UK. dsamuel@phls.nhs.uk
Abstract:
A simple, sensitive, specific and rapid procedure for isolating and typing polioviruses is described. Specimens are inoculated onto confluent monolayers of cell lines (Hep-2C, L20B or RD) seeded into microtitre plates. After 24-48 h, the infected cells are stained with monoclonal antibodies specific for poliovirus types 1,2,3 or a blend of the three antibodies followed by an anti-mouse IgG-horseradish peroxidase conjugate. On addition of substrate, infected cells stain an intense blue colour and are easily distinguished from uninfected cells by light microscopy. Poliovirus infection can be detected before the appearance of cytopathic effects (CPE). This Blue-Cell ELISA test was evaluated against conventional culture and seroneutralisation on a range of polio isolates and clinical specimens. The sensitivity and specificity of the Blue-Cell ELISA compared to neutralisation was 100% (87/87) on culture supernatants of poliovirus isolates sent to our reference laboratory for confirmation. All the poliovirus isolates were typed within 24 h of specimen inoculation using the new method compared to 6-10 days by conventional culture and neutralisation. The method proved to be more sensitive than conventional culture when clinical specimens were examined. Of 43 clinical specimens from which poliovirus had been previously isolated by various laboratories in the U.K., 30/43 (69.8%) were positive for poliovirus by the Blue-Cell ELISA compared to 29/43 (67.4%) by conventional culture and neutralisation. Neutralisation of specimens exhibiting CPE indicated that all of the polioviruses were correctly typed with the new method. CPE was not observed by conventional culture in any specimen that was negative in the Blue-Cell ELISA. There were no cross-reactions with a range of other enteroviruses.
Insights
A new Blue-Cell ELISA test rapidly detects and types polioviruses in 24 hours, outperforming traditional methods. This sensitive and specific assay identifies poliovirus infection before cytopathic effects appear.
Area of Science:
- Virology
- Immunology
- Diagnostic Assays
Background:
- Poliovirus detection and typing are crucial for disease surveillance and control.
- Conventional methods like cell culture and seroneutralization can be time-consuming and less sensitive.
Purpose of the Study:
- To develop and evaluate a simple, sensitive, specific, and rapid assay for isolating and typing polioviruses.
- To compare the performance of the new assay against established methods.
Main Methods:
- A Blue-Cell Enzyme-Linked Immunosorbent Assay (ELISA) was developed using microtitre plates with specific cell lines.
- Infected cells were stained with monoclonal antibodies against poliovirus types 1, 2, and 3, followed by an enzyme-conjugated secondary antibody.
- Detection of infection was achieved by a colorimetric reaction before the appearance of cytopathic effects (CPE).
Main Results:
- The Blue-Cell ELISA demonstrated 100% sensitivity and specificity compared to seroneutralization for poliovirus isolates.
- Typing of poliovirus isolates was completed within 24 hours, significantly faster than the 6-10 days required for conventional methods.
- The assay showed increased sensitivity in detecting poliovirus in clinical specimens compared to conventional culture and neutralization.
Conclusions:
- The Blue-Cell ELISA is a rapid, sensitive, and specific method for poliovirus detection and typing.
- This assay can identify poliovirus infection earlier than traditional methods, even before CPE is visible.
- The Blue-Cell ELISA offers a significant advancement for poliovirus surveillance and diagnostics.