Schmallenberg virus nucleocapsid protein monoclonal antibody, and preparation method thereof
A technology of monoclonal antibody and nucleocapsid protein, which is applied in the field of genetic engineering and immunity to achieve the effect of rich expression, strong specificity and strong antigen conservation
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[0052] 2 Preparation of immune splenocytes
[0053] After 3 days of booster immunization, aseptically collect splenocytes to prepare splenocyte suspension, the specific operation is as follows:
[0054] 1) Mice were killed by dislocation;
[0055]2) Soak the mouse in 75% alcohol for disinfection, cut its abdomen with sterile scissors, take out the spleen, put it in a sterile plate containing a small amount of culture solution (1640 culture solution) and put it on a stainless steel drying net. The needle core was ground into a cell suspension and counted;
[0056] 3) Collect the spleen cell suspension on ice, centrifuge to remove the supernatant;
[0057] 4) Wash the pellet again by centrifugation with fresh RPMI-1640 culture medium;
[0058] 5) Add fresh RPMI-1640 culture medium; count spleen cells.
[0059] 3 Recovery of myeloma cells
[0060] The myeloma cell SP2 / 0 was stored in a liquid nitrogen tank at -196°C, and it was sufficient to recover, proliferate, and passage...
Embodiment 3
[0105] Example 3 Anti-SBV N Protein Monoclonal Antibody Titer Determination——ELISA Detection
[0106] 1. Coating: The purified recombinant N protein was used as the detection antigen, the coating concentration was 2 μg / ml, 100 μL / well, overnight at 4°C, and washed 3 times with the washing solution.
[0107] 2. Blocking: add 150 μL / well blocking solution, wash 3 times after 2 hours at 37°C, and pat dry. Store in a 4°C refrigerator for later use.
[0108] 3. Add the sample to be tested:
[0109] 1) For serum / antibody / ascitic fluid titer detection, dilute the first well at 1:1000, then dilute with a gradient ratio of 1:2, incubate at 37°C for 30 minutes, wash the plate 4 times, and pat dry.
[0110]2) For cell supernatant detection, pipette 100 μL of cell supernatant, add it to the corresponding microplate, incubate at 37°C for 30 min, wash the plate 4 times, and pat dry.
[0111] 3) Add secondary antibody: take horseradish-enzyme-labeled goat anti-mouse IgG (IgG-specific seco...
Embodiment 4
[0117] Example 4 Western blot identification of anti-Schmallenberg virus nucleocapsid protein N monoclonal antibody (2C8)
[0118] 1 Purpose of the test
[0119] To verify whether the monoclonal antibody 2C8 can react with the recombinant N protein and the natural nucleocapsid protein (N) of SBV.
[0120] 2 test material
[0121] Cell line: BHK-21.
[0122] SBV strains: BH80 / 11-4, BH652 / 12-1, BH619 / 12, D512 / 12, D495 / 12-1, and D495 / 12-2; viral stock was 10 6 TCID 50 / mL.
[0123] HRP-goat anti-mouse IgG (product number P0447): purchased from Dako Company in Denmark.
[0124] 3 operation steps
[0125] 3.1 Preparation of protein samples
[0126] Cells infected by different strains of SBV and control cells (approximately 2.0×10 6 cells / bottle) were digested from the wall of the cell flask, transferred to a 15mL centrifuge tube with a pipette, centrifuged at 300×g at 4°C for 6min, poured off the cell supernatant, and resuspended the cell pellet with 2mL PBS. Store in a 4°...
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