CD15 - always consider your selection of the clone and format

CD15 is a surface marker for myeloid cells. It is also expressed with lower intensity on monocytic populations. In combination with CD34 it serves as a good marker for monitoring maturation process of myeloid cell populations.

From the previous experience in the laboratory, there has been a clone-related difference between IgG (W6D3) and IgM (HI98, MMA, MEM-158) isotypes of the monoclonal antibody. The respective clones distinguish individual populations with varying accuracy and stain index. Therefore, each is suitable for different applications. 4 different clones of monoclonal CD15 antibody conjugated into 3 different fluorochromes were tested.

As a pentamer forming structure, IgM clones tend to cluster, which results in loss of granulocytes due to aggregation and a higher off-scale signal (needs more adjustments namely titration and voltration). MMA clone seems to lead to particularly susceptible readouts with diminished granulocyte population. See Table 1 for quantification. (Raw data available upon request.) Neither MEM-158 nor HI98 resulted in such a strong aggregation. However, neither of the IgM clones allowed for a clear distinction between major populations (lymphocytes, monocytes, and granulocytes) based on their CD15 expression.

To conclude our experimental observations each isotype is suitable for a different application.
The IgG isotype results in a more accurate distinction among major populations present in whole blood. The best separation between granulocytes, monocytes and lymphocytes based on their CD15 expression was achieved using W6D3 clone (IgG) conjugated with FITC. See Figure 1 for detail.

In terms of monitoring the maturation process of myeloid cell lineage the best distinction with a clear path from immature through maturing to granulocytic and monocytic populations was achieved using the HI98 clone (IgM). See Figure 1 for detail.

Table 1: Overview of ratio of granulocyte population in a sample from one donor based on the antibody clone used for staining.
   
   

CD15	Clone	Isotype	% of granulocytes from Single cells
None FMO			75.9 75.7
BV785	W6D3	IgG1	75.6
FITC	W6D3	IgG1	77.1
FITC	MEM-158	IgM	75.0
V500	HI98	IgM	74.6
FITC	MMA	IgM	60.3

 

Fig. 1: A representative sample of different clones of CD15 antibody in various fluorochromes on all CD45 positive cells (left column). Expression of markers, CD15 and CD34 on a population gated as CD117 positive (right column).

 

Based on the shown results, please, always consider your selection of the clone of your CD15 antibody as well as its isotype and fluorochrome in order to accurately answer your scientific question. Each antibody is suitable for a slightly different application.

 

This blog was prepared for you by:

Petra Hadlová, Ph.D. candidate
CLIP-cytometry
Department of Paediatric Haematology and Oncology
Charles University and University Hospital Motol

 

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CD15 - clones W6D3, MEM-158 and MMA in different formats,

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