Human CTLA4 / CD152 ELISA Kit
- SKU:
- HUFI00481
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P16410
- Sensitivity:
- 0.094ng/ml
- Range:
- 0.156-10ng/ml
- ELISA Type:
- Sandwich
- Synonyms:
- CTLA4, Cytotoxic T Lymphocyte Associated Antigen 4, CD152, CD, CD152 antigen, CD28, celiac disease 3, CELIAC3, CTLA-4, cytotoxic T-lymphocyte protein 4, Cytotoxic T-lymphocyte-associated antigen 4, cytotoxic T-lymphocyte-associated protein 4, GRD4, G
- Reactivity:
- Human
- Research Area:
- Immunology
Description
system_update_altDatasheet - तकनीकी मैनुअलsystem_update_altMSDS - तकनीकी मैनुअल
Human CTLA4 / CD152 ELISA
CTLA4 / CD152 is a member of the immunoglobulin superfamily and encodes a protein which transmits an inhibitory signal to T cells. CTLA4 / CD152 contains a V domain, a transmembrane domain, and a cytoplasmic tail. Mutations in CTLA4 / CD152 have been associated with autoimmune diseases such as diabetes mellitus, graves disease, hashimoto thyroiditis, celiac disease, and systemic lupus erythematosus. The Assay Genie Human CTLA4/CD152 ELISA is a highly sensitive assay for the quantitative measurement of CTLA4/CD152 in serum, blood, plasma, cell culture supernatant and tissue samples.
| Product Name: | Human CTLA4 / CD152 ELISA Kit |
| Product Code: | HUFI00481 |
| Size: | 96 Assays |
| Alias: | CTLA4, Cytotoxic T Lymphocyte Associated Antigen 4, CD152, CD, CD152 antigen, CD28, celiac disease 3, CELIAC3, CTLA-4, cytotoxic T-lymphocyte protein 4, Cytotoxic T-lymphocyte-associated antigen 4, cytotoxic T-lymphocyte-associated protein 4, GRD4, GSE, ICOS |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human CTLA4 concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 0.094ng/ml |
| Range: | 0.156-10ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human CTLA4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CTLA4 in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CTLA4 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| CV(%): | Intra-Assay: CV<8% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
| Uniprot | P16410 |
| UniProt Protein Function: | CTLA-4: Inhibitory receptor acting as a major negative regulator of T-cell responses. The affinity of CTLA4 for its natural B7 family ligands, CD80 and CD86, is considerably stronger than the affinity of their cognate stimulatory coreceptor CD28. Genetic variation in CTLA4 influences susceptibility to systemic lupus erythematosus (SLE). SLE is a chronic, inflammatory and often febrile multisystemic disorder of connective tissue. It affects principally the skin, joints, kidneys and serosal membranes. SLE is thought to represent a failure of the regulatory mechanisms of the autoimmune system. Genetic variations in CTLA4 may influence susceptibility to Graves disease, an autoimmune disorder associated with overactivity of the thyroid gland and hyperthyroidism. Genetic variation in CTLA4 is the cause of susceptibility to diabetes mellitus insulin-dependent type 12 (IDDM12). A multifactorial disorder of glucose homeostasis that is characterized by susceptibility to ketoacidosis in the absence of insulin therapy. Clinical fetaures are polydipsia, polyphagia and polyuria which result from hyperglycemia-induced osmotic diuresis and secondary thirst. These derangements result in long-term complications that affect the eyes, kidneys, nerves, and blood vessels. Genetic variation in CTLA4 is the cause of susceptibility to celiac disease type 3 (CELIAC3). It is a multifactorial disorder of the small intestine that is influenced by both environmental and genetic factors. It is characterized by malabsorption resulting from inflammatory injury to the mucosa of the small intestine after the ingestion of wheat gluten or related rye and barley proteins. In its classic form, celiac disease is characterized in children by malabsorption and failure to thrive. 4 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Immunoglobulin superfamily; Membrane protein, integral Chromosomal Location of Human Ortholog: 2q33 Cellular Component: Golgi apparatus; perinuclear region of cytoplasm; integral to plasma membrane; plasma membrane; clathrin-coated endocytic vesicle; external side of plasma membrane Molecular Function:protein binding Biological Process: B cell receptor signaling pathway; negative regulation of T cell proliferation; positive regulation of apoptosis; negative regulation of regulatory T cell differentiation; T cell costimulation; negative regulation of immune response; immune response; negative regulation of B cell proliferation; response to DNA damage stimulus Disease: Autoimmune Lymphoproliferative Syndrome, Type V; Celiac Disease, Susceptibility To, 3; Diabetes Mellitus, Insulin-dependent, 12; Hashimoto Thyroiditis; Systemic Lupus Erythematosus |
| NCBI Summary: | This gene is a member of the immunoglobulin superfamily and encodes a protein which transmits an inhibitory signal to T cells. The protein contains a V domain, a transmembrane domain, and a cytoplasmic tail. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. The membrane-bound isoform functions as a homodimer interconnected by a disulfide bond, while the soluble isoform functions as a monomer. Mutations in this gene have been associated with insulin-dependent diabetes mellitus, Graves disease, Hashimoto thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, and other autoimmune diseases. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P16410 |
| NCBI GenInfo Identifier: | 27735177 |
| NCBI Gene ID: | 1493 |
| NCBI Accession: | P16410.3 |
| UniProt Secondary Accession: | P16410,O95653, Q0PP65, Q52MC1, Q53TD5, Q5S005, Q8WXJ1 Q96P43, Q9UKN9, A0N1S0, E9PDH0, |
| UniProt Related Accession: | P16410 |
| Molecular Weight: | 223 |
| NCBI Full Name: | Cytotoxic T-lymphocyte protein 4 |
| NCBI Synonym Full Names: | cytotoxic T-lymphocyte-associated protein 4 |
| NCBI Official Symbol: | CTLA4Â Â |
| NCBI Official Synonym Symbols: | CD; GSE; GRD4; ALPS5; CD152; CTLA-4; IDDM12; CELIAC3Â Â |
| NCBI Protein Information: | cytotoxic T-lymphocyte protein 4; CD152 isoform; celiac disease 3; insulin-dependent diabetes mellitus 12; cytotoxic T-lymphocyte-associated serine esterase-4; cytotoxic T lymphocyte associated antigen 4 short spliced form; ligand and transmembrane spliced cytotoxic T lymphocyte associated antigen 4 |
| UniProt Protein Name: | Cytotoxic T-lymphocyte protein 4 |
| UniProt Synonym Protein Names: | Cytotoxic T-lymphocyte-associated antigen 4; CTLA-4; CD_antigen: CD152 |
| Protein Family: | Cytotoxic T-lymphocyte protein |
| UniProt Gene Name: | CTLA4Â Â |
| UniProt Entry Name: | CTLA4_HUMAN |
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
| Step | Protocol |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells! |
| 2. | Aliquot 0.1ml standard solutions into the standard wells. |
| 3. | Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well. |
| 4. | Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells. |
| 5. | Seal the plate with a cover and incubate at 37 °C for 90 min. |
| 6. | Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2. |
| 7. | Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall. |
| 8. | Seal the plate with a cover and incubate at 37°C for 60 min. |
| 9. | Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash. |
| 10. | Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min. |
| 11. | Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. |
| 12. | Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color. |
| 13. | Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. |
| 14. | Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
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