The monoclonal antibody MR5D3 recognizes the Mannose Receptor (MR, also known as CD206), a member of the vertebrate C-type lectin family. MR is a pattern recognition receptor that is involved in both innate and adaptive immunity. The 175 kDa transmembrane protein consists of 5 domains: an amino-terminal cysteine-rich region, a fibronectin type II repeat, a series of eight tandem lectin-like carbohydrate recognition domains (responsible for the recognition of mannose and fucose), a transmembrane domain and an intracellular carboxy-terminal tail.The structure is shared by the family of multi lectin mannose receptors: the phospholipase A2-receptor, DEC 205 and the novel C-type lectin receptor (mannose receptor X). The MR recognises a wide range of gram positive and gram negative bacteria, yeasts, parasites and mycobacteria. Moreover, the MR has also been shown to bind and internalize tissue-type plasminogen activator.
MR’s are present on monocytes and dendritic cells (DC) and are presumed to play a role in innate and adaptive immunity, the latter via processing by DC. The expression of MR as observed in immunohistology is present on tissue macrophages, dendritic cells, a subpopulation of endothelial cells, Kupffer cells and sperm cells. The expression of MR on monocytes increases during culture and can be enhanced by cytokines such as IL-4. Labelling of MR expressing monocytes/macrophages increases with prolonged incubation time probably due to internalization of the MR-antibody-complex. Detection of the MR with anti-MR monoclonal antibody MR5D3 can substitute staining for mannose containing probes as labeled mannosylated BSA, a technique which is more cumbersome and less specific.
Flow cytometry, Frozen sections, Functional studies, Immuno assays, Immuno fluorescence, Immuno precipitation, Western blot
FC: 2% paraformaldehyde fixed cells were labeled with 10 µg/ml of mAb.
IHC-F:Sections fixed in ethanol or 2% paraformaldehyde were permeabilized with PBS/0.1% triton X100. After quenching and blocking mAb was added (10 µg/ml) and incubated for 1h
IA: mAb was used as detector at 10 µg/ml for 2h.
IP:MR was immunoprecipiated using antibody MR5D3 (10 µg/ml) and GammaBind Plus sepharose (Ref. 2)
W: Cleared lysates were run on 6% SDS-PAGE under nonreducing conditions. After transfer blot was stained with mAb ( 2 µm/ml).
FS: Antibody MR5D3 was used as a MR ligand to target MR+ dendtritic cells resulting in anti-rat IgG production (Ref. 6).
For immunohistochemistry, flow cytometry and Western blotting, dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50. For functional studies, dilutions have to be optimized in user’s experimental setting.