Rapid Detection Of Influenza A Virus Using A SPR Biosensor

H5N1 avian influenza has recently been recognized as an infectious disease that may pose a threat to animal and public health. Human cases of avian influenza A (H5N1) in Hong Kong in 1997 highlighted the public health importance of avian influenza prevention and control. Traditional methods for avian influenza virus detection and subtype identification are based on virus isolation in tissue culture or embryonated chicken eggs, such as real-time reversetranscriptase polymerase chain reaction (RT-PCR), NASBA and colloidal gold-based immunochromatographic assay. Each method has its own advantages, but limited by several unfavorable factors, such as the high cost, time consuming, low sensitivity, especially in the quantitative analysis aspect, as well as instantaneity and accuracy limitations in subtype identification. Compared with above methods, the advantages of SPR biosensor, such as real-time monitoring of antigen-antibody binding and dissociation reaction on biosensor surface, quantitative, rapid testing and label-free, are evident. This detection method could be taken as a complement to current methods, which can be used in AIV detection in clinical sample, chicken embryo allantoic fluid and cell culture.In this study, single-channel single-parameter SPR biosensor was used in rapid detection of avian influenza virus subtype H5. Rapid method for influenza A virus detection has been initially established based on the preliminary exploration, and subtype H5 and H9 of Influenza A was taken as an example.Four methods, assay through oriented antibody immobilization by immunoglobulin -binding staphylococcal protein A, competitive inhibition assay, antibody sandwich assay and direct detection assay, were compared during the course of AIV subtype H5 detection. Test results of each method were analyzed and compared. The result revealed that direct detection method has a good detection effect in lysed virus samples (lysed with 1% Triton X-100 for 90 min at room tempture) detection, Principle of the direct detection method is as follows: monoclonal antibodies specific to H5 hemagglutinin protein were immobilized on chip surface to capture virus particles passing over the sensing surface. Resultant changes on the surface were visualized directly in the detector. Existence and quantity of AIV in samples can be deduced according to the signal change. The detection range was established between 5ng/mL-500ng/mL. The specificity and stability of the antibody chip have been studied. The result revealed that the antibody chips had no binding with HSA, and also no binding with H9N2, another subtype of influenza A. This preliminary study had provided basis and reference for influenza A rapid detection using SPR biosensor, and proved that the chip has a good effect in H5 and H9 subtypes detection.