Paired antibodies for detecting novel coronavirus and their applications
A technology for detecting antibodies and pairing antibodies, which is applied in the field of immunoassays and can solve problems such as reducing the affinity of antibodies and N protein, lack of antibody combinations, and poor stability
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Embodiment 1
[0048] Example 1 Preparation of hybridoma cell lines
[0049] Hybridoma cell strain preparation: BL-21 (DE3) strain was transformed with pET-28a -N plasmid to express N protein solublely. BALB / c mice were subcutaneously immunized with 25 μg of antigen with Freund's complete adjuvant, and Freund's incomplete adjuvant was used for the second and third immunizations. Ten days after the third immunization, blood was collected by docking the tail, and the serum titer reached 1:640,000. 30 μg N protein was administered intraperitoneally for booster immunization. After 3 days, splenocytes were taken and SP2 / 0 myeloma cells were fused with PEG at a ratio of 5:1. 1 mL of preheated 50% PEG-1450 was left to stand for 1 min, shaken slowly in a water bath for 90 s, slowly added 15 mL of DMEM medium dropwise, and kept in a water bath at 37°C for 5 min. Add serum-free DMEM medium to 40 mL, centrifuge at 1000 rpm for 5 min and discard the supernatant. Add 40 mL of preheated HAT-containing ...
Embodiment 2
[0058] Example 2 Rapid detection of chemiluminescence
[0059]N24 antibody HRP labeling: (1) Weigh 25 mg of HRP and dissolve it in 1.25% glutaraldehyde solution, and let it stand overnight at room temperature. (2) The reacted enzyme solution was eluted with Sephadex G-25 chromatographic column with normal saline. The flow rate was controlled at 1 mL / min, and the brown effluent was collected. If the volume is greater than 5 mL, concentrate to 5 mL with PEG. Place in a 25mL small beaker and stir slowly. (3) Dilute 12.5 mg of the antibody to be labeled to 5 mL with normal saline, and add it dropwise to the enzyme solution while stirring. (4) Add 0.25mL of 1M pH9.5 carbonate buffer and continue to stir for 3 hours. (5) Add 0.25 mL of 0.2M lysine, mix well, and place at room temperature for 2 hours. (6) Add an equal volume of saturated ammonium sulfate dropwise under stirring, and place at 4°C for 1 hour. (7) Centrifuge at 3000rpm for half an hour, discard the supernatant. T...
Embodiment 3
[0064] Example 3 Preparation of test card
[0065] Antibody microsphere labeling: 300nm red carboxyl microspheres (Thermo) were washed with 50mM MES pH6.0 and diluted to 1mg / mL, and then ultrasonically dispersed (Ningbo Xinzhi), the ultrasonic program was 5%, ultrasonic 2s, interval 3s, ultrasonic 3min . EDC two-step method activates carboxyl microspheres, EDC, sulfo-NHS MES solution (5mg / mL) is added with 1% (1mg / mL) microspheres, NHS:EDC:-COOH is mixed at a ratio of 5:5:1, 30 ℃ rotation activation for 30min. After washing the microspheres with 50mM HEPES pH 7.4 coupling buffer by centrifugation at 13000rpm, add 80μg of N17 antibody per mg of microspheres, rotate and couple at 37°C for 2 hours, and add 5% BSA solution for blocking.
[0066] Take the PVC bottom plate, sample pad, conjugation pad, nitrocellulose membrane and absorbent paper, lap the sample pad, conjugation pad, reaction membrane and water absorption pad on the PVC bottom plate in sequence, and coat the N24 an...
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