Seemingly non-specific signal of anti-IgM antibodies

You may also have this experience. When staining human peripheral blood with anti-IgM antibody, you might see IgM signal not only on B cells, where you expect it, but also on a subset of CD19 negative lymphocytes, which looks like a non-specific signal, at least at a glance. We have analysed this phenomenon. Using mouse monoclonal antibodies CH2, SA-DA4, and MHM-88 we stained human peripheral blood samples and identified IgM signal on the surface of CD19 positive lymphocytes (B cells), and on a CD19 negative lymphocyte population, which turned out to be  CD56 positive cells (probably NK cells). Whereas B cells showed IgM signal also intracellularly, those IgM positive non-B cell lymphocytes had IgM only on their surface. It was a reasonable result, as IgM is not only expressed on B cells in monomeric form as a component of their antigen specific B cell receptor (BCR), but it is also secreted by them in a 900 kDa pentameric form to the serum, where it serves as an efficient complement binder. IgM antibodies make up about 10% of all serum antibodies. There is, however, also second mechanism of IgM attachment to the B cell surface, it is via the IgM-specific Fc receptor (FcµR), and this receptor can be expressed also on NK cells and on a subset of T cells. Thus the source of seemingly non-specific signal of anti-IgM antibodies makes sense now. It was a specific signal of IgM attached to the surface of FcµR-expressing CD19 negative cells.
 
Here we have some details regarding our results. After pilot experiments that included only cell surface staining, we analysed also intracellular IgM expression. To this end we stained normal patient blood samples with an excess of anti-Hu IgM APC (clone CH2; cat. no. 1A-320-C100), then we permeabilized the blood cells and stained them intracellularly using anti-Hu IgM FITC (clone CH2; cat. no. 1F-320-C100). Our results showed that B cells are stained by anti-Hu IgM antibody both extra- and intracellularly, while within a subset of CD19 negative cells the IgM signal was only on the surface (Fig. 1, 2). In other experiments we identified overlay of CD19 negative IgM positive lymphocytes and of CD19 negative CD56 positive lymphocytes (Fig. 3, 5, 6), and we confirmed that the staining pattern of anti-IgM antibody clone CH2 was independent on the presence of anti-CD56 antibody (Fig. 4). Furtheremore, comparing the results done with the clone CH2 and with two other anti-human IgM clones, SA-DA4 and MHM-88, we saw the same staining patterns (Fig 5, 6).


 

Fig. 1: Cell surface staining profile of human lymphocytes detected using anti-human IgM (CH2) APC antibody and anti-human CD19 (LT19) PE-Cy™7 antibody. Note IgM signal both on CD19 positive and on CD19 negative cells.


 
 
Fig. 2: Intracellular IgM (CH2) signal was identified only on CD19 positive and not on CD19 negative cells.
 


 
Fig. 3: Visualization of all lymphocytes (black), CD19 positive B cells (red), CD19 negative IgM positive lymphocytes (blue) and CD19 negative CD56 positive lymphocytes (green). IgM was stained only on the cell surface in this experiment. There was an overlay of CD19 negative IgM positive cells and of CD19 negative CD56 positive cells, so that all CD56 positive cells were IgM positive on their surface.
 
 

 
Fig. 4: Anti-IgM (CH2) binding to CD56 positive cells was independent on the presence of anti-CD56 antibody. On the left, there are lymphocytes detected in a sample containing anti-CD56 FITC, on the right, there are lymphocytes in a sample without any anti-CD56 antibody.

 
 
 
Fig. 5: Anti-human IgM clone CH2 (on the left) has the same lymphocyte staining pattern as other two anti-IgM clones SA-DA4 (upper right), and MHM-88 (bottom right), both in the same concentration as CH2. CD19 negative IgM positive cells are indicated in blue, CD19 positive cells (B cells) are indicated in red. 

 


Fig. 6: Anti-human IgM clone CH2 (on the left) stains the same CD19 negative CD56 positive cells as other two anti-IgM clones SA-DA4 (upper right), and MHM-88 (bottom right), both in the same concentration as CH2. CD19 negative IgM positive cells are indicated in blue, CD19 positive cells (B cells) are indicated in red. 
 
 
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