Preparation and application of total antibody ELISA (enzyme linked immunosorbent assay) kit for detecting fever accompanied by thrombocytopenia syndrome virus
A kit and total antibody technology, which is applied in the field of preparation of ELISA kits for detecting fever with thrombocytopenia syndrome virus total antibodies, can solve the problem of inability to assess the protection effect of vaccines on the level of previous infection in the population and the like
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Embodiment 1
[0015] Embodiment 1: Expression and purification of rNP protein
[0016] The inactivated Neobunia virus JS-2007-001 strain with the accession number CCTCCV201211 was used to prepare a viral RNA template using a conventional QIAamp RNA viral RNA mini kit (Qiagen, Germany).
[0017] The following primers were used as amplification primers:
[0018] SEQ ID No. 1:
[0019] Primer1 5`-ggatccgcatgcATGTCAGAGTGGTCCAGGATTG-3`;
[0020] SEQ ID No.2:
[0021] Primer2 5`gccaagcTTACAGATTCCTGTAAGCAGCAGCAG-3`;
[0022] Utilize the conventional Invitrogen One step RT-PCR kit (Invitrogen, USA) to amplify the open reading frame of the New Bunia virus NP protein; use conventional molecular biology methods to insert the reading frame into the position of the bacterial expression plasmid pQE30 (Qiagen, Germany) Point between SphI and HindIII to obtain bacterial expression plasmid pQE30-NP. The plasmid was routinely transformed into E. Coli M15 strain (Qiagen, Germany), induced to express 6His...
Embodiment 2
[0023] Example 2: Preparation of rNP protein-coated ELISA reaction plate.
[0024] Purified NP protein was diluted to (10 μg / mL) with 0.05M pH 9.6 carbonate coating buffer. Routinely add the diluted NP protein solution into the wells of the reaction plate, 100 μL per well, place at 2-8°C for coating for 18-20 hours, discard the liquid in the wells, wash the plate 3-5 times, add enzyme stabilizer ( Shandong, Taitian and Biology) 150-200 μL. React at 2-8°C for 4-6 hours. Discard the enzyme stabilizer in the well, dry the coated plate by freeze-drying method, seal the reaction plate in a sealed bag, and store it at 2-8°C for later use.
Embodiment 3
[0025] Example 3: HRP-rNP protein preparation.
[0026] rNP was labeled by modified sodium periodate method (Basic Medical Immunology Experiment, Jin Boquan, Li Enshan. Beijing World Book Publishing House, 1990). Dissolve 5mg of HRP in 0.5mL of distilled water, add 0.5mL of freshly prepared 0.06M NalO4 aqueous solution, mix well and place in a refrigerator at 4°C for 30 minutes, take out and add 0.5mL of 0.16M ethylene glycol aqueous solution, place at room temperature for 30 minutes, then add Purified NP protein aqueous solution 1mL, mix well and put in a dialysis bag, slowly stir and dialyze with 0.05M, pH9.5 carbonate buffer solution in a 4°C refrigerator for 6 hours to combine, then suck it out, add NaBH4 solution (5mg / mL) 0.2mL, put it in a 4°C refrigerator for 2 hours, add the above-mentioned conjugate mixture into an equal volume of saturated ammonium sulfate solution, put it in a 4°C refrigerator for 30 minutes, and centrifuge, and dissolve the obtained precipitate in...
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