Human CTLA-4 and B7 Inhibitor Screening ELISA Kit (B7-1 and B7-2)

Catalog Number:

F24D54

Background:

Human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a member of the CD28-B7 superfamily and acts as a co-inhibitory receptor for T-cell activation. B7 protein, including B7-1 (CD80) and B7-2 (CD86), expressed on antigen‐presenting cells binds to CTLA-4 on T cells to prevent T cells from multiplying and attacking. This prevents the immune system from overreacting and harming healthy tissue. This also brings the strategy for the development of anti-tumor molecules such as antibody or small molecule medicine to block this interaction to fight cancer.

Applications:

This kit is developed to screen the tested molecules, such as antibodies or other molecules, against CTLA-4 that block the interaction between CTLA-4 and B7-1 or B7-2 of your choice. The assay detects any molecules or antibodies in serum/plasma or purified antibody (monoclonal & polyclonal) that inhibit CTLA-4 binding to B7-1 or B7-2 in vitro. The test is both species and isotype independent. It is for research use only.

Description:

This kit was validated by a known monoclonal antibody – Lpilimumab. The binding between CTLA-4 and B7-1 or B7-2 is blocked by this antibody in a dose-dependent manner, see figures.

Ordering:

F24D54-1 for blocking test between CTLA-4 and B7-1

F24D54-2 for blocking test between CTLA-4 and B7-2

Biosafety Level:

BSL-1

Assay Principles:

This kit is developed for a blocking ELISA format with a 96-well plate pre-coated with CTLA-4 protein. The whole process should take about 2.5 hours. The samples or provided positive/negative controls will be added to bind to CTLA-4. The positive control will be captured on the plate by CTLA-4, while the negative control will remain in the supernatant and get removed during washing.  A labeled B7-1 or B7-2 (provided) will be added to the plate. After detecting and reading at 450 nm, the positive control wells generate a lower reading as it blocks the binding between CTLA-4 and B7 while negative control wells generate a higher reading. The absorbance of the sample is inversely dependent on the titer of the antibodies or molecules blocking this interaction between CTLA-4 and B7.

The kit consists of 96-well detachable plate with CTLA-4 pre-coated, Positive control, Negative control, labeled B7 (B7-1 or B7-2 of your choice), TMB and dilution/wash/stop buffer.

Brief protocol (detailed protocol is provided with the product):

1. Detach wells from plate based on your samples and keep at room temperature. Dilute your samples, positive and negative controls in sample dilution buffer and add them to wells (0.1 ml/well). Incubate at 37°C for 1 hour or room temperature for 1.5 hour. Wash wells 4 times by wash solution (0.3 ml/well).
2. Add labeled B7 (100 µl/well). Incubate at room temperature for 1 hour. Wash wells as mentioned.
3. Add TMB to each well (100 µl /well) and incubate at room temperature for 10-30 minutes.
4. Stop the substrate reaction by adding stop solution (100 µl /well). Absorbance (OD) is calculated as the absorbance at 450 nm. The OD Value inversely reflects the amount of antibodies or molecules blocking this interaction between CTLA-4 and B7.

Storage and shipping condition:

Shipment with room temperature. The unopened kit is stable for 6 months from the date of manufacture when stored at 2°C to 8°C. Avoid light. The shelf life is 60 days from the date of opening.