Mouse igg elispot basic kit alp

In order to further verify that the vaccine polypeptide can stimulate the body to produce specific anti-E. g antibody, the Enzyme-Linked Immunospot (ELISPOT) experiment was carried out. The ELISPOT plate was coated with vaccine polypeptide (5 μg/ml) in coating buffer overnight at 4-8°C. The plate was washed and blocked with 100 μl of RPMI 1640 medium (Sigma Aldrich, St. Louis, UA) containing 10% fetal calf serum (FCS) (Sigma) for 1h at 37°C. The plate was washed by PBS containing 0.05% Tween 20. The mice splenocytes suspension (2×10⁵) and the vaccine polypeptide were added in plates, and every samples were added to three repeated wells. The plate was incubated for 20h at 37°C under 5% CO₂. The biotinylated detecting antibody (anti-IgG) and Streptavidin-ALP was added. The plate was incubated at 37°C for 30 minutes and the BCIP/NBT (mlbio) solution was added and incubated for color development. Last, antibody secreting cell (ASC) spot were counted using an ELISPOT automatic plate reader (AID Elispot Reader, AID, Germany). The Mouse IgG ELISpot BASIC kit (ALP) (3825-2A, Mabtech, Stockholm, Sweden) was adopted for the ELISPOT experiment.

The Enzyme-Linked Immunospot (ELISPOT) experiment was performed for evaluating the specific B-cell response in mice. First, the rSMEV vaccine protein solution (10 μg/mL) was coated on the ELISPOT plate and incubated overnight at 4 °C. Then the plate was washed for 5 times by Microplate washer (Thermo Fisher, FI-01620 Vantaa, Finland) and blocked with 200 μl RPMI-1640 medium (Sigma Aldrich, St. Louis, UA) containing 10% fetal calf serum (FCS) (Sigma) for 30 minutes at 37 °C. After the plate was washed by PBS containing 0.05% Tween 20, the vaccine protein and mice splenocytes suspension prepared (2×10⁵) were added into the ELISPOT plate3. Three repeat wells were set for each sample and the plate was incubated for 20 hours in a 37 °C humidified incubator with 5% CO2. After the plate was washed for 5 times, the biotinylated detecting antibody (anti-mouse IgG) was added and the plate was incubated for 2 hours at room temperature. After the plate was washed, the Streptavidin-ALP was added and incubated at room temperature for 1 hour. The BCIP/NBT solution (MlBio) was added and incubated for chromogenic reaction. The plate was rinsed and dried, the antibody secreting cell (ASC) spot were counted using EliSpot Reader (AID, D 72479, Germany). In this experiment work, the Mouse IgG ELISpot BASIC kit (ALP) (3825-2A, Mabtech, Stockholm, Sweden) was adopted for the ELISPOT experiment.