Test paper strip for detecting novel coronavirus antibodies, and preparation method and application thereof
A technology of test strips and antibodies, applied in the direction of viruses/bacteriophages, biochemical equipment and methods, viruses, etc., can solve the problems of complex operation and long detection process
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Embodiment 1
[0077] The preparation and purification of embodiment 1 SARS-CoV-2-NP
[0078] Take the inactivated SARS-CoV-2 strain, prepare the viral RNA template, and amplify the open reading frame of the SARS-CoV-2 protein; and insert the bacterial expression plasmid pET-28(a) to obtain the bacterial expression plasmid pET-28(a )-NP. The plasmid was routinely transformed into E.CoLi BL21 strain, and protein expression was induced in LB broth at 37°C with 50 μg / ml kanamycin and 1 mM isopropyl-β-D-thiogalactopyranoside. Cultured pellets containing recombinant proteins were resuspended in chromatography binding buffer and sonicated. The cell lysate was centrifuged, and the supernatant was used to load the nickel ion affinity column. After the column was thoroughly washed with binding buffer, the recombinant 6His-tagged NP protein was eluted from the column with elution buffer, and the purified protein was analyzed by SDS-PAGE and visualized by Coomassie brilliant blue staining.
[0079] ...
Embodiment 2
[0080] Example 2 ELISA detects the effect of recombinant expression protein binding virus-specific IgM
[0081] Coat the ELISA plate with NP full-length protein, NP-NT and NP-NC protein respectively, 100ng / well, overnight at 4C. After washing, block with 5% skimmed milk powder, overnight at 4°C. Add 100 μL of sample diluent to each well, then add 1 μL of serum to be tested, mix well, incubate at 37°C for 30 minutes, wash 5 times, add mouse anti-human IgM-HRP (μ chain specific) to detect virus specificity IgM, incubate at 37°C for 30 minutes, wash 5 times, add TMB+H 2 o 2 Substrate, 100 μL / well, let stand at room temperature for 5 minutes, then add stop solution 50 μL / well. The OD value was measured at a wavelength of 450 nm.
[0082] The result is as figure 2 As shown, it can be seen that the detection effect of NP-NT is the best, so NP-NT is used as the T line coating antigen in the present invention.
Embodiment 3
[0083] The preparation of the colloidal gold detection card of embodiment 3 SARS-CoV-2
[0084] 1. Materials and reagents
[0085] 1.1 Immunochromatographic materials
[0086] Glass cellulose membrane, nitrocellulose membrane, absorbent paper, adhesive bottom plate, and rapid detection plastic card were all purchased from Shanghai Jieyi Biotechnology Co., Ltd.
[0087] 1.2 Preparation of main reagents
[0088] HAuCl 4 Solution: 1g HAuCl 4 Dissolve in 100mL ultrapure water, filter through a 0.22μm filter, wrap in tin foil, and store in the dark at 4°C.
[0089] Trisodium citrate solution: 1g of trisodium citrate powder, add ultrapure water to 100mL, dissolve and filter with a 0.22μm filter, subpackage and store at 4°C for later use.
[0090] BSA solution: Weigh an appropriate amount of BSA powder according to the percentage, add 100mL of phosphate buffer solution with pH=7.4, fully dissolve, and let stand. Store at 4°C for later use, but only for short-term storage. It i...
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