Cluster Of Differentiation 5 (CD5) is a cluster of differentiation found on a subset of IgM-secreting B cells called B-1 cells, and also on T cells. B-1 cells have limited diversity of their B-cell receptor due to their lack of the enzyme terminal deoxynucleotidyl transferase (TdT) and are potentially self-reactive. CD5 serves to mitigate activating signals from the BCR so that the B-1 cells can only be activated by a very strong stimuli (such as bacterial proteins) and not by normal tissue proteins. CD5 was used as a T-cell marker until monoclonal antibodies against CD3 were developed. In humans, the gene is located on the long arm of chromosome 11. T cells express higher levels of CD5 than B cells. CD5 is upregulated on T cells upon strong activation. Cluster Of Differentiation 19 (CD19) has a remarkably high affinity for CD5, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human CD5 and recombinant mouse CD19. Briefly, biotin-linked CD5 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to CD19-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µl stop solution to the wells and read at 450 nm immediately. The binding activity of recombinant human CD5 and recombinant mouse CD19 was shown in Figure 1, the EC50 for this effect is 10.6 ug/mL.