Comparison of four anti-human CD16 clones specificity

CD16 (FcγRIII) is a 50-65 kDa glycoprotein serving as a low affinity IgG receptor and a receptor for oligomeric IgE. Human NK cells, macrophages, activated monocytes, mast cells and γ/δ T cells express CD16a, a type I transmembrane protein, which associates with TCR ζ chain and FcεRI γ chain to transduce signals. This isoform is responsible for antibody-dependent NK cell cytotoxicity. Polymorphism in amino acid 158 was described, which affects its affinity for IgG Fc. CD16b is a GPI-linked monomeric receptor constitutively expressed on neutrophils. It is involved in their activation and induction of a proadhesive phenotype. Importantly, the affinity of CD16 for single IgG Fc is much lower than the affinity of anti-CD16 IgG for CD16, as CD16 serves in the immune system to detect immune complexes and opsonized cells, not individual immunoglobulins.
 
For customers who plan to use an anti-CD16 antibody it is important to know which isoforms the particular mAb clone detects and whether (and how) it depends on CD16 polymorphism. Hence we decided to compare four anti-CD16 clones from our portfolio regarding their reactivity with blood cell populations gated using innate immunity panel and adaptive immunity panel.
 
All four tested antibody clones 3G8, LNK16, MEM-154, and MEM-168 showed comparable and specific signal when detecting CD16b. Concerning CD16a, the antibody MEM-154, which needs 158V polymorphic variant, lacked CD16a signal in one subject (obviously with 158F variant), whereas in another subject its CD16a signal was comparable with those of other three Mab clones.
 
 
 
Fig. 1: Comparison of four anti-CD16 clones (3G8, LNK16, MEM-154, and MEM-168) concerning their reactivity with particular blood cell populations in human buffy coats of subject A and subject B. All four antibody clones detected CD16a on NK cells, as well as CD16b on neutrophils with comparable signal in subject A, whereas MEM-154, which is sensitive to CD16a-158V/F polymorphism (it needs presence of V at amino acid 158), reacted only with CD16b on neutrophils, but not with CD16a on NK cells and monocytes in subject B, indicating that this person had 158F polymorphism. For gating antibodies description and gating strategy see Appendix below.
 
 
 
 
 
 
 Fig. 2: Granulocytes were detected by mAb clone MEM-154 both in subject A and subject B, whereas monocytes and NK cells were detected only in subject A, indicating that subject A has 158V, and subject B has 158F variant of CD16a.                
 
 
 

Appendix:

 
 
Links to anti-CD16 clones used in this study:
3G8
LNK16
MEM-154
MEM-168
 
Gating strategy for innate immunity panel:

 

 
 
Gating strategy for adaptive immunity panel:

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