Figure 1. B7-H4 protein is expressed in SK-BR-3 cells but absent in H1299 cells, as detected using the PathScan® Total B7-H4 Sandwich ELISA Kit #14520. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blot using B7-H4 (D1M8I) XP® Rabbit mAb #14572 is shown in the bottom figure.
Figure 2. The relationship between protein concentration of lysates from SK-BR-3 cells and the absorbance at 450 nm as detected by the PathScan® Total B7-H4 Sandwich ELISA Kit #14520 is shown. SK-BR-3 cells (85% confluence) were harvested and then lysed.
|Product Includes||Volume||Solution Color|
|B7-H4 Rabbit mAb Coated Microwells||96 tests|
|B7-H4 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
PathScan® Total B7-H4 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of B7-H4. A B7-H4 Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, B7-H4 protein is captured by the coated antibody. Following extensive washing, a B7-H4 Mouse Detection mAb is added to detect the captured B7-H4 protein. A HRP-linked, anti-mouse antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of total B7-H4.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Total B7-H4 Sandwich ELISA Kit detects endogenous levels of B7-H4 protein as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
B7 homolog 4 (B7-H4, VTCN1) is a member of the B7 family of cell surface ligands that regulate T cell activation and immune responses (1-3). B7-H4 protein contains two extracellular Ig-like V-type domains, a transmembrane domain, and a short, two amino acid intracellular domain (3). The B7-H4 protein is shown to inhibit T cell activation, proliferation, and cytokine production (1,4,5). Although B7-H4 mRNA is widely expressed, B7-H4 protein is restricted to antigen presenting cells and B cells (1). The B7-H4 protein is also found in several tumor types, including ovarian cancer and breast cancer (6). Research studies indicate that B7-H4 protein is present on the surface of ovarian tumor cells, and that targeted inhibition of B7-H4 using recombinant antibodies restores T cell activation pathways. These studies suggest some potential therapeutic value in blocking B7-H4 function and restoring T cell function in cancer patients (7,8).
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