Monitoring Antibody Secreting Cells with the B cell ELISPOT

B cell ELISPOTB cell

U-CyTech offers B cell ELISPOT kits for detection of human, monkey and mouse antibody secreting cells (ASCs). 

The B cell ELISPOT assay has been developed to enumerate ex vivo antigen-specific B cells on basis of antibody production at the single cell level. The assay is useful in different fields of biomedical research including vaccine development, infection, drug treatment and autoimmune diseases. For example, the number of ASCs following an immunization can be used to predict the magnitude and longevity of protection against a specific infectious disease.
 

Activation of memory B cells

During an immune response, two types of cells are created, ASCs and memory B cells. Whereas ASCs are short-lived, memory B cells have a long lifespan and are responsible for long lasting immunity against a specific antigen. Because of low frequencies in peripheral blood, slow proliferation and lack of antibody secretion, memory B cells need first to be activated and expanded in vitro  by a 2-6 day polyclonal stimulation before they can be enumerated in the B cell ELISPOT assay.
 

B cell ELISPOT method

U-CyTech’s B cell ELISPOT is based on the same solid-phase immunoenzymatic principle as the T cell ELISPOT. It also has a high sensitivity and easy performance, making the B cell ELISPOT assay eminently well suited to monitor B cell responses at the single cell level. The ASCs are enumerated in the ELISPOT plate coated with antibodies (directed to a specific class of immunoglobulin [Ig] e.g. IgG) or an antigen of interest. Finally, spot-forming cells are detected by labeled antibodies directed to the Ig class of choice.
 

Examples of the B cell ELISPOT assay
Human IgG B cell ELISPOT      Mouse IgG1 B cell ELISPOT
The picture on the left demonstrates the results of tetanus-specific IgG secreting B cells produced by 5x104 human PBMC/well in 7 hours. In this assay, silver staining reagents for spot detection and a transparent polystyrene-bottomed plate were used. To activate the memory B cells, cells were preincubated in vitro for 5 days in the presence of a polyclonal B cell stimulus.
The picture on the right is an example of total IgG1 secreting B cells produced by 1x105 mouse (Balb/c) splenocytes/well in 20 hours. Spots were visualized by enzymatic staining on a PVDF-membrane bottomed plate. Activation of memory B cells was accomplished by an in vitro preincubation period of 2 days in the presence of a polyclonal B cell stimulus.

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