Subunit influenza vaccine-specific antibody-secreting plasma cells in bone marrow were detected by ELISPOT assay. Multiscreen HTS-IP filter 96 well plates (Millipore, Billerica, MA) were coated with antigen (1 μg/ml) overnight at 4°C, and next day, 106 bone marrow cells (collected from femur and tibia) without red blood cells in complete RPMI 1640 cell culture medium were added to each well and incubated overnight in a 37°C incubator with 5% CO2. Secreted anti-hemagglutinin-IgG1 and IgG2a were then detected by alkaline phosphatase (AP)-conjugated goat anti-mouse IgG1 and IgG2a antibodies (Southern Biotech), and the antibody spots were developed by Vector Blue AP substrate (Vector Laboratories, Burlingame, CA). An ELISPOT reader was used to image and count the spots in the well.
Vector blue ap substrate
Manufactured by Vector Laboratories
Sourced in United States
Vector Blue AP substrate is a chromogenic substrate for alkaline phosphatase. It is used to detect and visualize the presence of alkaline phosphatase in various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using vector blue ap substrate
1
Quantifying Influenza Vaccine Antibodies
2
Quantifying Antigen-Specific T-Cell Responses
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Briefly, 96-well MultiScreen filter plates (Merck Millipore, USA) were coated overnight at 4°C with 5 μg/ml capture antibodies for anti-IL-2, anti-IL-10 and anti-IFN-γ (MABTECH, Sweden). 1x106 pooled lymph node and spleen cells were added to the wells and incubated overnight at 37°C, in the presence or absence of 0.1 μM of BDC2.5 peptide. After removal of the cells, plates were washed and 1 μg/ml biotinylated anti-IL-2, anti-IL-10 and anti-IFN-γ detection antibodies (MABTECH) added for 1 hour at room temperature. Plates were washed and spots detected by incubation with streptavidin alkaline phosphatase (Jackson ImmunoResearch, USA), followed by Vector Blue AP substrate (Vector Laboratories, USA). Number of spots was calculated with an ImmunoSpot analyzer and software (ImmunoSpot, USA).
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