There are two basically different protocols for the B cell ELISPOT assay both of which are required to determine the precise frequency of memory B cells in peripheral blood cell preparations.
1. With preincubation
For monitoring memory B cells, an in vitro preincubation period ranging from 2 to 6 days is necessary to expand and activate the B cells using a particular polyclonal stimulus.
2. Without preincubation
Stimulation of B cells is not required for the enumeration of antigen-specific B cells that have recently been activated in vivo (for instance 3-9 days after vaccination).
To determine the number of antigen-specific B cells, the wells of the ELISPOT plate are coated with an antigen of interest, post-coated with a blocking agent and sub-sequently incubated with a cell preparation. Thereafter, the assay is completed as des-cribed below.
When the total number of antibody secreting plasma cells (ASCs) (irrespective of its antigen specificity) is determined, the wells of the ELISPOT plates are coated with a coating antibody (provided with the kit) that specifically binds to the antibodies released by the ASCs. After blocking, cells are incubated for a defined length of time in the wells of the ELISPOT plate.
After incubation, cells and debris are washed away. Areas on the bottom of the wells where the antibodies (released by the ASCs) are captured by the immobilized antigen or antibody, are detected with biotinylated anti-Ig (sub)class specific antibodies followed by incubation with enzyme-labeled streptavidin or gold-labeled anti-biotin antibodies.
The last step in the B cell ELISPOT assay is addition of a substrate yielding a colored zone ('spot'), which reveals the site of antibody secretion (footprint of an individual cell). These footprints represent either the antigen-specific B cells (in antigen-coated wells) or the total number of ASCs (in antibody-coated wells).
The protocols determining the antigen-specific B cells and total number of ASCs are usually performed in parallel. Since the stimulation step expands the cells, the number of antigen-specific B cells is usually compared to the total number of ASCs to obtain an accurate estimate of the frequency of antigen-specific B cells present.