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| ID | Components | Size |
| EP117-C01 | High-bind Plate | 1 plate |
| EP117-C02 | Human CTLA-4 | 10 μg |
| EP117-C03 | Biotinylated Human B7-2 | 65 μg |
| EP117-C04 | Anti-CTLA-4 Neutralizing Antibody | 20 μg |
| EP117-C05 | Streptavidin-HRP | 10 μg |
| EP117-C06 | Coating Buffer | 12 mL |
| EP117-C07 | 10xWashing Buffer | 50 mL |
| EP117-C08 | Blocking Buffer | 50 mL |
| EP117-C09 | Substrate Solution | 24 mL |
| EP117-C10 | Stop Solution | 7 mL |
Background/背景
Cytotoxic T lymphocyte associated ANTIgen-4 (CTLA-4), also known as CD152, is a kind of leukocyte differentiation antigen and a transmembrane receptor on T cells, sharing B7 ligand with CD28. The binding of CTLA-4 with B7 induces T cells to be non-reactive and participates in the negative regulation of immune response. The recombinant CTLA-4 Ig can effectively and specifically inhibit cellular and humoral immune responses in vivo and in vitro, and has significant therapeutic effects on transplant rejection and various autoimmune diseases with very low toxicity. It is considered as a promising new immunosuppressive drug at present.
Application/应用
This kit is developed for screening for inhibitors of human CTLA-4 binding to human B7-2.
It is for research use only.
Reconstitution/重构方法
Please see Certificate of Analysis for details of reconstitution instruction and specific concentration.
Storage/存储
The unopened kit is stable for 12 months from the date of manufacture if stored at 2°C to 8°C. The opened kit should be stored per components table. The shelf life is 30 days from the date of opening.
Assay Principles/原理
This inhibitor screening ELISA kit is designed to facilitate the identification and characterization of new CTLA-4 pathway inhibitors. The assay takes advantage of our in house-developed binding of biotinylated human B7-2 to immobilized human CTLA-4 in a functional ELISA assay, and employs a simple colorimetric ELISA platform. Briefly, we provide you with a human Biotinylated B7-2 protein, a human CTLA-4 protein, an anti-CTLA-4 neutralizing antibody (as method verified Std.), and Streptavidin-HRP reagent.
Your experiment will include 4 simple steps:
a) Coat the plate with human CTLA-4.
b) Add your molecule of interest to the tests.
c) Add human B7-2-Biotin to bind the coated human CTLA-4.
d) Add Streptavidin-HRP followed by TMB or other colorimetric HRP substrate.
Finally, the ability of your compound to inhibit B7-2: CTLA-4 binding will be determined by comparing OD readings among different experimental groups.
Typical Data/典型数据
B7-2 [BIOTINYLATED]: CTLA-4 BINDING IN THE ABSENCE OF INHIBITORS
Immobilized human CTLA-4 protein at 0.2 μg/mL (100 μL/well) can bind biotinylated human B7-2 with a linear range of 0.01-20 μg/mL when detected by Streptavidin-HRP. Background was subtracted from data points before curve fitting (QC tested).
Protocol
INHIBITION OF B7-2 [BIOTINYLATED]: CTLA-4 BINDING BY ANTI-CTLA-4 NEUTRALIZING ANTIBODY
Serial dilutions of Anti-CTLA-4 Neutralizing antibody (Catalog # EP117-C04) (1:1 serial dilution, from 10 μg/mL to 0.01μg/mL) was added into CTLA-4: Biotinylated B7-2 binding reactions. The assay was performed according to the protocol described below. Background was subtracted from data points prior to log transformation and curve fitting (QC tested).
Protocol
