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Human CTLA-4 Quantikine HS ELISA Kit

Bio-Techne includes R&D Systems | Catalog # HSCT40

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HSCT40

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Key Product Details

Assay Length

4.0 hours

Sample Type & Volume Required Per Well

Cell Culture Supernates (100 µL), Cell Lysates (100 µL), Serum (100 µL), EDTA Plasma (100 µL), Heparin Plasma (100 µL), Urine (100 µL)

Sensitivity

0.13 pg/mL

Assay Range

0.781-50 pg/mL (Cell Culture Supernates, Cell Lysates, Serum, EDTA Plasma, Heparin Plasma, Urine)

Product Summary for Human CTLA-4 Quantikine HS ELISA Kit

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.

Product Specifications

Measurement

Quantitative ELISA

Detection Method

Colorimetric - 450 or 490nm (TMB or AP Substrate)

Conjugate

HRP

Reactivity

Human

Specificity

Natural and recombinant human CTLA-4.

Cross-reactivity

Interference

No significant interference observed with available related molecules.

Precision

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, EDTA Plasma, Heparin Plasma, Serum, Urine

Intra-Assay Precision Inter-Assay Precision
Sample 1 2 3 1 2 3
n 20 20 20 20 20 20
Mean (pg/mL) 5.53 15.6 32.1 5.32 14.7 31.2
Standard Deviation 0.191 0.409 0.528 0.331 0.851 1.98
CV% 3.5 2.6 1.6 6.2 5.8 6.3

Recovery for Human CTLA-4 Quantikine HS ELISA Kit

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

Sample Type Average % Recovery Range %
Cell Culture Media (n=4) 105 102-108
Cell Lysis Buffer (n=1) 76 75-78
EDTA Plasma (n=4) 90 79-97
Heparin Plasma (n=4) 93 84-100
Serum (n=4) 94 85-102
Urine (n=4) 103 100-107

Linearity

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.

CTLA-4 ELISA Sample Linearity

Scientific Data Images for Human CTLA-4 Quantikine HS ELISA Kit

CTLA-4 ELISA Standard Curve

Human CTLA-4 High Sensitivity ELISA Standard Curve

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: CTLA-4

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).

Long Name

Cytotoxic T-lymphocyte-associated Molecule 4

Alternate Names

CD152, CTLA4

Entrez Gene IDs

1493 (Human); 12477 (Mouse); 403696 (Canine); 102115124 (Cynomolgus Monkey)

Gene Symbol

CTLA4

Additional CTLA-4 Products

Product Documents for Human CTLA-4 Quantikine HS ELISA Kit

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Human CTLA-4 Quantikine HS ELISA Kit

For research use only

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