| The continuous spread of highly pathogenic avian influenza virus (AIV) subtype H5N1 is threatening the poultry industry and human health worldwide. Currently, there are several analytical methods for the detection of H5 virus were proposed, such as virus isolation (VI), standard reverse transcription-PCR (RT-PCR), real-time RT-PCR, enzyme-linked immunosorbent assay (ELISA), and so on. However, these assays have their limitations, such as time-consuming, low sensitivity, technically demanding and so on. Therefore, a simple, rapid and sensitive detection assay for the early diagnosis of H5 is required to lower the chances of spread and reduce the risk of development into an epidemic. This research include the following three parts:1. Preparation of polyclonal antibody angainst HA protein of AIV subtype H5 and IgG extractionAccording to the designed immunization schedule, two SPF rabbits were chosen and immunized with the E.coli expressed HA protein. When the antibody titer of agarose-gel precipitation(AGP) get to the level that we want, we can obtain the rabbits'serum. After these sera were purified by n-Caprylic acid-ammonium sulphate and gel column G-200 respectively, we successfully got the highly depurated rabbit anti-AIV IgG.2. Preparation of QDs-rabbit anti-AIV IgG probeThe water-soluble CdTe QDs were linked to the rabbit anti-AIV IgG using the coupling reagents ethyl-3-(dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The couple condition was optimized, and the molar QD:IgG ratio in the reaction was discussed. The resonance light scattering (RLS) was used for detection of antigen-antibody recognition reactions, and results showed that the probe retained the activity of antibody and was suitable for immunoassay.3. Development of sFLISA for the H5N1 subtype AIVIn this research, a novel sandwich fluorescence-linked immunosorbent assay (sFLISA) for the determination of H5 subtype AIV was developed basd on the McAb and QDs-rabbit anti-AIV IgG, which both against HA of H5 subtype AIV. The developed sFLISA supplied an effective approach to early diagnosis of AIV subtype H5N1 in fields.Under the optimal conditions, the sFLISA allowed for H5N1 viral antigen determination in a linear range of 8 ng/mL～510 ng/mL with the limit of detection (LOD) of 0.15 ng/mL, which was lower than commercial ELISA. Moreover, in comparison with virus isolation (VI) for 103 clinic samples, the sFLISA was correlated with VI for 92.2%, the sensitivity and specificity of sFLISA were found to be 93.6% and 91.1% respectively. Results showed that the sFLISA was specific and sensitive for the H5N1 sbtype AIV.The developed sFLISA supplied a novel approach to rapid and sensitive detection of AIV subtype H5N1, and provided significant reference for QDs in the application of animal-borne diseases.
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