ELISpot and FluoroSpot are considered the gold standard method for finding rare, antigen-specific B cells. With this method you can assess both antibody secreting cells and memory B cells, as well as these cells’ antigen-specificity, isotype, and subclass.
B cell ELISpot and FluoroSpot assays are powerful tools in measuring immunoglobulins directly upon secretion. Commonly, these assays are used to directly enumerate antibody-secreting cells (ASCs) and long-term memory B cells. Because of their extreme sensitivity, the methods enable identification of rare antigen-specific B cells in as few as one in 250,000 cells – a difficult challenge for other methods such as flow cytometry. The B cell FluoroSpot assay utilizes the sensitivity of ELISpot, but in a multiplex format using fluorescence for detection. This allows for analysis of multiple antigens, immunoglobulin isotypes, or subclasses in a single well.
The main applications of B cell ELISpot and FluoroSpot include the detection of B cell responses to infections and responses elicited by vaccination, but it’s up to you which direction to explore!
Measure ASC frequency or memory B cell magnitude
ASCs can be directly assessed with our kits from in vivo stimulation following vaccination or infection. Memory B cells require ex vivo stimulation to differentiate into ASCs for assessment with ELISpot/FluoroSpot. We’ve optimized and validated the process with all the required stimulation reagents (R848 + IL-2) included in our kits. Read how to use the B cell StimPack in our tutorial on memory B cell stimulation.
B cell ELISpot
The B cell ELISpot assay counts ASCs using a sandwich assay of capture and detection antibodies or antigen. Total Ig B cell ELISpot (3) captures and detects all ASC antibodies and is often performed in parallel with an antigen-specific assay (1 or 2) to determine the ratio of antigen-specific cells. The antigen-specific assay may be performed in two different ways: either with reverse ELISpot (2), using a tagged antigen for detection, or traditional ELISpot (1) with the antigen coated on the plate and using a detection antibody. With both total and antigen-specific ELISpot, every spot represents one single cell.
Reverse ELISpot has several advantages for detection of antigen-specific cells. The reversed assay produces more distinct spots without any risk for antigen denaturation and it also dramatically reduces the amount of antigen required for each assay.
B cell ELISpot step-by-step
B cell FluoroSpot
FluoroSpot combines the sensitivity of ELISpot with the capacity to multiplex the assay. Multiple immunoglobulin isotypes, subclasses, and antigen-specificities of either memory B cells or ASCs can be assessed simultaneously utilizing fluorophore conjugated detection. This enables the analysis of unique and rare B cell profiles from a single well. Just like with ELISpot, the FluoroSpot assay builds on the sandwich assay, but instead of enzymatic reactions for detection, fluorophore conjugated detection antibodies or antigens are utilized to visualize B cell responses.
B cell FluoroSpot step-by-step
B cell FluoroSpot applications
Utilizing reverse B cell FluoroSpot allows for the detection of up to four different antigens in a single well. This saves both on cells and antigen. Antigens can be directly conjugated with fluorophores or recombinantly expressed with peptide tags and then detected with an anti-tag fluorophore conjugated antibody.
The use of coating antigen or capture antibodies followed by specific fluorophore-conjugated detection antibodies enables the detection of antigen-specific or total isotypes and immunoglobulin subclasses from a single well.
Multiple isotypes or subclasses