Time-resolved fluorescent immunoassay kit for highly pathogenic avian influenza virus h5n1
A time-resolved fluorescence, avian influenza virus technology, applied in the biological field, can solve the problems of inability to detect the virus at an early stage, complex operation requirements, and high cost, and achieve the goal of reducing the risk of H5N1 infection, reducing anxiety and panic, and reducing economic losses. Effect
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Embodiment 1
[0046] Example 1 Preparation of anti-human avian influenza virus H5N1 single chain antibody
[0047] (1) Construction of anti-human avian influenza virus H5N1 single-chain antibody phage library
[0048] 1) Isolation of total RNA: Human total RNA was extracted from 10mL whole blood of a patient who recovered from avian influenza H5N1 using a blood RNA extraction kit (Qiagen Corp).
[0049] 2) Synthesize the first cDNA strands of human IgG light and heavy chain genes: the RNA extracted in the above step is used as a template. In order to increase the specificity of amplification, reverse transcription primers (HuIgG1-4H1FOR, HuCkFOR and HuCλFOR) are used for specific amplification. cDNA first strand of human IgG1 constant region and light chain kappa, lambda constant region.
[0050] The sequence of the above-mentioned reverse transcription primer is:
[0051] HuIgG1-4H1FOR: 5'-GTC CAC CTT GGT GTT GCT GGG CTT-3' (SEQ ID NO: 9);
[0052] HuCkFOR: 5'-AGA CTC TCC CCT GTT GAA GC...
Embodiment 2
[0084] Example 2 Time-resolved fluorescent immunoassay kit for highly pathogenic avian influenza virus H5N1
[0085] The time-resolved fluorescent immunoassay kit for H5N1 contains the following components:
[0086] 1) Anti-H5N1 single-chain antibody
[0087] The anti-H5N1 single-chain antibody is selected from the single-chain antibody A1 (SEQ ID NO: 1), A10 (SEQ ID NO: 2), B9 (SEQ ID NO: 3) and F11 (SEQ ID NO: 4) One of.
[0088] 2) Enzyme-linked plates coated with antibodies
[0089] Dilute the coating antibody B9 (A1, A10, or F11) to 1-5 μg / ml with 50 mmol / L, pH 9.6 carbonate buffer, then add 100 μl / well to each well of the Elisa plate overnight at 4°C, pour off the coating solution, add blocking solution, 200 μl / well, block overnight, pour off the coating solution, wash and pat dry, and store in a vacuum at -20°C.
[0090] 3) Eu 3+ labeled antibody
[0091] Add 0.5mg of antibody F11 (A1, A10, or B9 can also be selected) into a centrifuge tube with a filter membrane ...
Embodiment 3
[0100] Example 3 Time-resolved fluorescent immunoassay method for highly pathogenic avian influenza virus H5N1
[0101] The present invention adopts the kit described in Example 2 to quantitatively detect H5N1 virus. The detection operation steps are: add 25 μl / well reference standard or sample to be tested to the antibody-coated enzyme-linked plate, and then add 200 μl / well for analysis Buffer solution, incubate with shaking for 1h, wash with washing solution 4 times, then add Eu diluted with analysis buffer at a volume ratio of 1: (50-100) 3+ Labeled antibody 200μl / well, shaking incubation 1h,. The washing solution was washed 6 times, and finally 200 μl / well of enhancement solution (purchased from Perkin Elmer) was added to shake for 5 minutes, and then detected on a time-resolved fluorescence detector.
[0102] The effect detection of the kits prepared in the above examples is performed below.
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