Skip to main content
. Author manuscript; available in PMC: 2016 Oct 1.
Published in final edited form as: J Immunol Methods. 2015 Jun 19;425:79–87. doi: 10.1016/j.jim.2015.06.012

Figure 3.

Figure 3

(A) Shown is a reducing (SDS) gel stained with Coomasie Blue with a protein ladder in the left column (Precision plus Protein Kaleidescope Standards, Bio-Rad, Cat # 1610375) and a band corresponding to KIR2DL3-Fc at 50.7kDa in the right column. The gel was loaded with 15 μl of KIR2DL3-Fc protein (100μg/ml).

(B) KIR2D-Fc bound to IgG coated beads (Bangs Laboratories) are stained with KIR specific antibodies to assess their integrity. Representative flow cytometry plots showing staining of KIR2DL1 (left panel, red) and KIR2DL3-Fc (right panel, blue) fusion proteins with EB6 and DX27 antibodies respectively. (C) Titrations of the binding of KIR2D to beads coated with HLA class I molecules. Each of three KIR2DL1-Fc fusion proteins distinguished by substitutions at residue 70 bind to HLA-C*06:02 with different avidities. Both mutant and wild type KIR2DL1 show saturated binding at concentrations greater than 100μg/ml. KIR2DL3-Fc binds to three HLA class I allotypes (HLA-B*73:01, HLA-C*03:04 and HLA-C*16:01) with different avidities. The binding of KIR2DL3-Fc to each allotype becomes saturated at concentrations above 100μg/ml. (D) KIR2DL1-Fc binds to HLA-C2 bearing allotypes but not to HLA-C1, HLA-A or HLA-B allotypes. KIR2DL3-Fc binds to HLA-C1 allotypes and to HLA-C2 allotypes with lower avidity. KIR2DL3-Fc also binds to two HLA-B allotypes that encode the C1 epitope. (E) KIR-Fc fusion proteins are amenable to long-term storage at −80°C. The binding of KIR2DL1-Fc stored at −80°C for 12 months is compared to 2DL1-Fc stored at 4°C.