CD38 is a multifunctional transmembrane glycoprotein expressed on the surface of many immune cells, including plasma cells, activated T and B lymphocytes, natural killer (NK) cells, and some myeloid cells. It also exists in a soluble form in bodily fluids. Here we present our comparison data of two anti-human CD38 clones commonly used in Flow cytometry with a potentially alternative clone EM5.
Functionally, CD38 acts as both a receptor and an enzyme. As an ectoenzyme, it catalyzes the conversion of NAD⁺ into cyclic ADP-ribose (cADPR) and ADP-ribose, which play important roles in calcium signaling and cellular activation. CD38 is also involved in cell adhesion and signal transduction.
Clinically, CD38 is an important marker in hematology. It is highly expressed on multiple myeloma cells and is targeted by therapeutic monoclonal antibodies, such as daratumumab and isatuximab. Its expression is also used as a prognostic marker in chronic lymphocytic leukemia (CLL).
While isatuximab binds to a distinct CD38 epitope and therefore does not interfere with the binding of most commonly used diagnostic anti-CD38 clones, daratumumab recognizes the same epitope as HB7 and HIT2. This epitope overlap leads to competitive binding and may prevent these clones from detecting CD38 on patient cells, posing challenges for flow cytometric diagnostics in daratumumab-treated patients. Our objective is to add an anti-CD38 clone with different epitope specificity in our portfolio, to provide universal anti-CD38 cocktail for identification CD38 positive plasma cells in both daratumumab and isatuximab treated patients. Here we examined clone EM5.
At first, we examined specificity of three anti-human CD38 clones – HIT2, HB7 and EM5 on human peripheral whole blood of two donors using innate immunity and adaptive immunity panels (based on Kužílková D. et al., Front Immunol. 2022;13:827898). We did not observe any difference in specificity of these clones (see Fig. 1 and 2).
Next, the staining profiles and other staining parameters of three anti-human CD38 clones were analyzed on peripheral whole blood of two healthy blood donors. Antibody conjugates with PE were titrated at 5 concentrations (8; 2; 0,5; 0,125; 0,03125 µg/ml).
Five-point titration curves for MFI and Stain index of all three clones are presented in Fig. 3 (positive – monocytes) and Fig. 4 (positive - CD19 positive CD38 positive B cells). As negative population, CD19 negative CD38 negative were used for all Stain Index enumerations.
In the last part, we examined, whether EM5 recognize the same epitope as HIT2 and HB7. Therefore, we conducted a competition analysis for these three clones. The competing (first) antibody (15 µg/ml) was added to blood sample 20 minutes prior the competed (second) antibody (1 µg/ml). The test design is summarized in Table 1 and results are shown in Fig. 5.
According our data, EM5 provides slightly lower MFI and shall be used in higher concentrations compared to HIT2 and HB7 clones. As EM5 has the same specificity and recognizes the same epitope of CD38 as HIT2 or HB7, it is not the clone to fulfill out goal. Therefore, we will keep looking for a suitable alternative to clones HIT2 and HB7 with different epitope specificity. If you are using, or know of, an anti-CD38 clone that remains effective in detecting plasma cells following daratumumab administration, please consider sharing this information with us. Available formats of HB7 antibody Available formats of HIT2 antibody Would you like to have our antibodies in other formats? Let us know by e-mail or use our FORM!
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