Human CTLA-4 Quantikine HS ELISA Kit

The Quantikine^TM HS Human CTLA-4 Immunoassay is a 4.0 hour solid-phase ELISA designed to measure human CTLA-4 in cell culture supernates, cell lysates, serum, plasma, and urine. It contains CHO cell-expressed recombinant human CTLA-4 and antibodies raised against the recombinant protein. Results obtained using natural human CTLA-4 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural human CTLA-4.

Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision

Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians

The recovery of human CTLA-4 spiked to levels throughout the range of the assay was evaluated.

To assess the linearity of the assay, samples containing and/or spiked with high concentrations of human CTLA-4 were serially diluted with calibrator diluent to produce samples with values within the dynamic range of the assay.

Scientific Data

CTLA-4 plays a role in immune self-tolerance, which is important for avoiding autoimmune diseases. Unfortunately, this mechanism can be co-opted in order to suppress immune responses in the context of cancer. CTLA-4's highly studied role in the T cell response has been well described (4). CTLA-4 was originally identified as a gene that was specifically expressed by cytotoxic T lymphocytes. However, CTLA-4 transcripts have since been found in both T helper type 1 and 2 (Th1 and Th2) cells, and CD4+ and CD8+ T cell clones. CTLA-4 is a negative costimulatory molecule that is upregulated over the course of 2-3 days in T cells in response to T cell receptor (TCR) engagement with peptide major histocompatibility complex (pMHC) on antigen presenting cells. CTLA-4 recruitment to the immunological synapse is positively correlated with TCR signal strength. CTLA-4 outcompetes CD28, a positive costimulatory molecule, for the ligands B7-1 (CD80) and B7-2 (CD86) in order to attenuate T cell activation. CTLA-4 has a much higher affinity and avidity for B7-1 and B7-2 than CD28. CTLA-4 and CD28 are structurally homologous molecules that are members of the immunoglobulin (Ig) gene superfamily. CTLA-4 and CD28 are both expressed on the cell surface as disulfide-linked homodimers or as monomers. Unlike CTLA-4, CD28 expression is constitutive on the surfaces of 95% of CD4+ T cells and 50% of CD8+ T cells and is down regulated upon T cell activation. CTLA-4 negative co-stimulation suppresses T cell activation, thereby facilitating immune evasion. CTLA-4's effects occur via cell intrinsic and cell extrinsic mechanisms. Cell intrinsic mechanisms include inhibition of protein translation, recruitment of phosphatases, activation of ubiquitin ligases, inhibition of cytokine receptor signaling, and inhibition of lipid domain formation at membrane of T cells. Cell extrinsic mechanisms include stimulating the release of the suppressive indoleamine (2,3) deoxygenase (IDO), modulation of regulatory T cell function and the aforementioned competitive inhibition of CD28 costimulatory signaling (5). 

CTLA-4 is a well-known immune checkpoint target. The human monoclonal CTLA-4 antibody Ipilimumab was approved by the food and drug administration (FDA) to treat melanoma in 2011 (4). Since then, it has been approved for colorectal cancer in children 12 and older, hepatocellular carcinoma, NSCLC, and renal cell carcinoma. The Ipilimumab binding epitope overlaps with the B7 binding domain and blocks the interaction via steric hinderance of B7(4). Ipilimumab allows for B7-CD28 positive costimulatory signaling, which is an important signal for T cell activation (5).