Question: Can I titrate the ratio of biotinylated antigen to BEAM Conjugate to improve the detection of antigen-positive cells in a Barcode Enabled Antigen Mapping (BEAM-Ab) experiment?
Answer: In the BEAM-Ab assay, antigen-positive cells are identified by flow cytometry using the PE fluorophore on the BEAM-Ab Conjugate. If optimal separation between PE positive and negative populations is not achieved, it may be possible to titrate the ratio of biotinylated antigen to BEAM Conjugate
During the development of the BEAM-Ab assay, our team utilized well-characterized models of antigen positivity to optimize the amount of biotinylated antigen and BEAM Conjugate to add when generating the BEAM-Ab Assembly. The recommended amount of antigen depends on the molecular weight of the antigen and can be calculated using the BEAM-Ab Workbook (CG000597).
It is possible that further optimization of antigen quantity may be beneficial in certain situations. If the antigen molecular weight or concentration were not determined accurately, you may inadvertently be adding too much or too little antigen when generating the BEAM-Ab Assembly. It is also possible that optimization of antigen quantity may be beneficial depending on the sample type, antigen type, antigen quality, biotinylation efficiency, and expected percentage of antigen-positive cells.
Adding an appropriate amount of biotinylated antigen is important for the following reasons:
- Adding too much antigen may lead to excess antigen that does not bind to the BEAM Conjugate to generate a BEAM-Ab Assembly. This unlabeled antigen (without PE) may compete with the BEAM-Ab Assembly for binding to target cells. Theoretically, this may hinder the detection of the antigen positive (PE positive) population.
- Adding too little antigen may lead to excess BEAM Conjugates that are not bound to any antigens. These “empty” BEAM Conjugates may contribute to increased background PE signal in certain sample types.
If the antigen quantity recommended in our BEAM-Ab documentation does not lead to optimal separation between PE positive and negative populations in your experiment, you may consider performing titration experiments to increase or decrease the amount of biotinylated antigen used to prepare the BEAM-Ab Assembly. For example, to test lower antigen amounts, you could reduce the amount of antigen by 2-fold, 5-fold and 10-fold, while keeping the amount of BEAM Conjugate constant, and keeping the total volume of BEAM Assembly constant, as shown in the example below.
Products: Single Cell Immune Profiling, Barcode Enabled Antigen Mapping (BEAM)