Human CTLA-4 Quantikine HS ELISA Kit Summary
Sample Type & Volume Required Per Well
Cell Culture Supernates (100 uL), Cell Lysates (100 uL), Serum (100 uL), EDTA Plasma (100 uL), Heparin Plasma (100 uL), Urine (100 uL)
0.8 - 50 pg/mL (Cell Culture Supernates, Cell Lysates, Serum, EDTA Plasma, Heparin Plasma, Urine)
Natural and recombinant human CTLA-4.
< 0.5% cross-reactivity observed with available related molecules.< 50% cross-species reactivity observed with species tested.
No significant interference observed with available related molecules.
The QuantikineTM 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
Cell Culture Supernates, Cell Lysates, Serum, EDTA Plasma, Heparin Plasma, Urine
The recovery of human CTLA-4 spiked to levels throughout the range of the assay was evaluated.
||Average % Recovery
|Cell Culture Media (n=4)
|Cell Lysis Buffer (n=1)
|EDTA Plasma (n=4)
|Heparin Plasma (n=4)
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.
Human CTLA-4 High Sensitivity ELISA Standard Curve
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Preparation and Storage
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Cytotoxic T-Lymphocyte-Associated molecule-4 (CTLA-4), also known as cluster of differentiation
152 (CD152) is located on band q33-q34 on chromosome 2 in humans, and band C of chromosome
1 in mice (1). CTLA-4 has over 100 single nucleotide polymorphisms (SNPs) associated with
autoimmunity and cancer. SNPs in the CTLA-4 promoter have been correlated with a variety of
cancers including breast cancer, cervical cancer, colorectal cancer, head and neck cancer,
hepatocellular cancer, melanoma, non-small-cell lung carcinoma (NSCLC), renal cancer and more.
The 223 amino acid (aa) protein product consists of several domains including: a 36 aa leader
peptide, a 116 aa extracellular Ig V-like ligand binding domain, a 37 aa transmembrane domain and
a 34 aa cytoplasmic domain. The cytoplasmic domain contains tyrosine motifs, at Y201 VKM and
Y218 FIP. Several intracellular proteins bind Y201 VKM including: phosphatidyl-inositol 3 kinase
(PI3-K), Src homology 2 domain containing protein tyrosine phosphatase (SHP-2), the serine
threonine phosphatase PP2a and clathrin adaptor proteins activator proteins (AP1, AP2).The mature
type 1 transmembrane glycoprotein has a native molecular weight of 33-37 kDa (2,3).
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).
Cytotoxic T-lymphocyte-associated Molecule 4
Entrez Gene IDs:
1493 (Human); 12477 (Mouse); 403696 (Canine); 102115124 (Cynomolgus Monkey)
CD; CD152 antigen; CD152; CD152IDDM12; CD28; celiac disease 3; CELIAC3; CTLA4; CTLA-4; cytotoxic T-lymphocyte antigen 4; cytotoxic T-lymphocyte protein 4; Cytotoxic T-lymphocyte-associated antigen 4; cytotoxic T-lymphocyte-associated protein 4; cytotoxic T-lymphocyte-associated serine esterase-4; GRD4; GSE; ICOS; ligand and transmembrane spliced cytotoxic T lymphocyte associated antigen 4