Rapid one-step antigen detection method for human bocavirus
Abstract number: O249
Koskinen J.O., Söderlund-Venermo M., Hedman K., Österback R., Heikkinen T., Waris M., Allander T., Vainionpää R., Soini A.E.
Objectives: Most research with human bocavirus, a recently found respiratory pathogen, has been done by molecular biology (polymerase chain reaction, PCR). The results have been ambiguous because the virus has often been found in co-infection with other viruses, and also in clinically healthy subjects. It has been proposed that, for bocavirus, antigen detection could better indicate the aetiology than qualitative nucleic acid detection. We have developed a rapid antigen detection test for the virus.
Methods: The one-step test for bocavirus VP2 antigen is based on a separation-free two-photon excitation fluorometry (ArcDia TPX assay technique). The assay protocol is simple; the swab sample is dissolved in sample buffer, and the solution is dispensed (20 ml) onto a 384-well microtitre plate (containing the reagents in dry form) for incubation and automated quantitative measurement. The immunoassay applies microspheres as solid-phase carriers of purified bocavirus-specific polyclonal antibodies. The virus antigens concentrate onto the solid-phase which is probed in real-time with fluorescently labelled antibody reagents. Strong positive samples are reportable in 15 minutes, while low positive and negative samples are reported in 2 hours. The performance of the method was studied with recombinant human bocavirus-like particles (VP2), and purified respiratory pathogens (Group A streptococci, Streptococcus pneumoniae, and influenza A and B, respiratory syncytial, metapneumo, adeno, and parainfluenza 13 viruses).
Results: Analytical detection sensitivity of the method (lowest limit of detection, 0-control + 3SDs) was 3 ng/ml, dynamic concentration range was three orders of magnitude, and intra-assay imprecision was 510%. Cross-reactions with the other respiratory pathogens were not found.
Conclusion: The new method enables rapid detection of bocavirus antigens. The new test is very easy to perform in comparison to standard ELISAs. The analytical sensitivity of the method is expected to allow analysis of clinical samples. The sensitivity of the antigen detection test could be significantly increased by the use of monoclonal antibodies (10100 fold). Our future objectives include increasing the detection sensitivity, and analysis of clinical samples in order to study the correlation of antigen detection and the clinical aetiology.
|Session name:||19th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Helsinki, Finland, 16 - 19 May 2009|
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