Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay
Transcrição
Antibody-secreting B cells, or ASCs are differentiated B cells that secrete antibody isotypes, including IgG and IgM, to ensure protection against pathogens.
To perform a functional assessment of ASCs in vitro, begin with the enzyme-linked immunosorbent spot assay. Take an ELISpot plate containing an activated polyvinylidene fluoride membrane at the base.
Incubate the wells with polyclonal antibody fragments exclusively containing Fab regions without the antibody's Fc region. Each Fab region has a binding site for specific antibody isotypes — IgG or IgM, enabling the identification of both isotypes.
During incubation, the antibody fragments attach to the membrane, leading to immobilization.
Overlay the wells with blocking solutions to prevent non-specific binding. Seed the plate with activated antibody-secreting B cells, and incubate. The ASCs secrete different isotypes, including IgG and IgM, which bind to the membrane-bound antibody fragments.
Wash the plate with a buffer to remove unbound secreted antibodies. Treat some wells with enzyme-conjugated detection antibodies for IgM and others with detection antibodies for IgG, and incubate.
The detection antibodies bind to their respective isotypes, forming immunocomplexes.
Add colorimetric substrates to all the wells, which interact with the enzymes and produce purple-colored spots on the membrane. Add a stopping solution to stop the reaction.
The colored spot produced in each well indicates the presence of the specific antibody's isotype, correlating with the secretory activity of B cells.
