Compositions and methods for modulating lair signal transduction
A technology for compositions and immunomodulators, applied in chemical instruments and methods, drug combinations, DNA/RNA fragments, etc., which can solve problems such as unclear functions
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Embodiment 1
[0496] Example 1: LAIR antibodies and their heavy and light chain sequences
[0497] Materials and methods
[0498] Mouse anti-human LAIR-1 monoclonal antibody
[0499]Mice were immunized with soluble human LAIR-1 (soluble LAIR-1 refers to the extracellular domain of LAIR-1) fused to murine G2a Fc (SEQ ID NO: 10). Mice were challenged 2 weeks later with the same immunogen. Mice received a third dose of antigen after two weeks. Three days after the last boost, mouse splenocytes were harvested and resuspended in RPMI supplemented with 10% FBS and glutamine, and later fused to form hybridomas.
[0500] RACE
[0501] RACE (Rapid Amplification of cDNA Ends) to identify heavy and light chains was performed according to the following protocol: (1) mRNA denaturation, (2) cDNA synthesis, (3) 5' RACE reaction, (4) analyzed PCR results (on agar Glycogel to visualize the amplified DNA fragments - correct antibody variable region DNA fragments should have a size between 500-700 base...
Embodiment 2
[0792] Example 2: Purification of LAIR-2Fc
[0793] Methods and materials
[0794] LAIR-2hIgG1 (hereinafter referred to as LAIR-2Fc) was produced by using the CHOK1SV KO parental line transfected with the Lonza GS vector. This cell line was used to express the lead candidate LAIR-2hIgG1 (native IgG1) and the mutated Fc version LAIR2-hlgG1Fc (L145A / L146A). LAIR-2Fc was purified by protein A chromatography and analyzed by SDS-PAGE ( Figure 6A ) and size exclusion chromatograms to assess purity ( Figure 6B ). LAIR-1Fc was prepared similarly to the control, and LAIR-2Fc was purified by size exclusion chromatography and visualized using SDS PAGE.
[0795] result
[0796] Figure 6A is an SDS-PAGE gel showing LAIR-2Fc under reducing and non-reducing conditions. Figure 6B is a chromatogram showing a single main peak at 38.550 minutes. The data confirm the expected size of LAIR-2Fc, as well as the high level of purity of the LAIR-2Fc protein used in the studies describe...
Embodiment 3
[0797] Example 3: Binding of LAIR-2Fc to Collagen
[0798] Materials and methods
[0799] K562AML cell lines with stable expression of collagen 17 or controls lacking collagen 17 were stained with 1 ug of LAIR-2Fc and LAIR-1Fc, incubated on ice for 30 min, and then washed in FACS buffer (PBS+1% FBS) Cells were then stained with 0.05ug anti-hlgG-PE for 30 minutes on ice. Cells were washed, resuspended in FACS fixation buffer (3% paraformaldehyde in PBS) and evaluated by flow cytometry.
[0800] result
[0801] LAIR-2Fc was assessed by its ability to bind the endogenous transmembrane ligand collagen 17 expressed on the surface of K562 cells ( Figure 7A with 7B ) and LAIR-1Fc ( Figure 7C with 7D )Feature. SDS-PAGE analysis of LAIR-2Fc and LAIR-1Fc was used to assess the purity of the proteins used in this and subsequent studies ( Figure 7E with 7F ). For all data presented here, a purity of greater than 95% for LAIR-2Fc and LAIR-1 Fc was standard.
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