Binding data showing the reactivity of rhKIR2DL2-Fc chimera bound to four HLA-C allotypes. The KIR2DL2 protein is able to distinguish between allotypes (e.g HLA-C*03:02) that bind strongly and those that are less well recognized (e.g HLA-C*04:01). Results are shown relative to the absolute amount of HLA class I antigen present on each bead as determined by binding to the pan HLA class I antibody W6/32.
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Our lab investigates how the diversity of innate immune system genes impacts immune system function. Specifically, we study the clonal diversity arising from differential expression of NK cell receptors (e.g. killer-cell immunoglobulin like receptors – KIR) both in individuals and in human populations. Investigation of specific receptor ligand interactions that alter NK cell function have been complicated by the inherent diversity of the system. However, this research has been facilitated by the development of soluble KIR-Fc fusion proteins in which the extracellular portions of the KIR molecule are fused to the Fc portion of a human IgG molecule. Such molecules may be used in an assay that detects direct binding to transfected cell lines expressing a single HLA class I allotype or to beads coated with a single HLA class I antigen.
Detection of KIR2DL2 reactive HLA-C molecules expressed on primary cells, transfected cells or beads coated with single HLA class I antigens.
Beads coated with single HLA class I antigens, cells transfected with single HLA class I molecules (e.g 721.221) or primary cells.
The rhKIR2DL2-Fc chimera is reconstituted at 100ug/ml in PBS. 100ul of reconstituted protein (10ug total) is added to beads coated with single HLA class I antigens and incubated for 60min at 4°C. The beads are then washed and stained with a PE-conjugated anti-human IgG1-Fc antibody (One Lambda; cat # is LS-AB2) and incubated for a further 60 min at 4°C. Samples are analyzed on a Luminex 100 reader platform that determines the unique identification of a specific bead and the fluorescence of the bound secondary antibody.
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This highly purified recombinant protein binds specifically to HLA class I allotypes that recognize KIR2DL2 (HLA-C). The high reported purity of the chimera protein is reflected in the fact that there is little or no background binding to other HLA-A or HLA-B allotypes in the panel. Within HLA-C there is a variable recognition of allotypes, with for example, HLA-C*04:01 being recognized at a lower level than other HLA-C allotypes.
The level of background binding seen is likely to be higher with primary cells or transfected cells as the Fc portion of the recombinant construct has a tendency to show non-specific binding to other proteins. This binding is reduced in the setting of a purified HLA class I allotype annealed onto a bead surface.
Product Name: Recombinant human KIR2DL2-Fc/CD-158b1 Chimera, 50ugSupplier Page