Human CTLA4 ELISA Kit (HUEB0355)- High Sensitivity

Product Name:	Human Cytotoxic T-lymphocyte protein 4 (CTLA4) ELISA Kit
SKU:	HUEB0355
Size:	96T
Target:	Human Cytotoxic T-lymphocyte protein 4 (CTLA4)
Synonyms:	Cytotoxic T-lymphocyte-associated antigen 4, CTLA-4, CD152, CD152
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.78-50ng/mL
Sensitivity:	0.33ng/mL

Intra CV:

4.1%

Inter CV:

7.8%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	83-93%	107-116%	107-117%	87-99%
EDTA Plasma(N=5)	83-93%	101-110%	103-112%	103-112%
Heparin Plasma(N=5)	97-107%	111-119%	102-114%	94-103%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	93	87-99
Plasma	95	89-101

Function:

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.

Uniprot:	P16410
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Cytotoxic T-lymphocyte protein 4
Sub Unit:	Homodimer; disulfide-linked. Binds to CD80/B7-1 and CD86/B7.2.
Research Area:	Development Biology
Subcellular Location:	Cell membrane Single-pass type I membrane protein Exists primarily an intracellular antigen whose surface expression is tightly regulated by restricted trafficking to the cell surface and rapid internalisation;.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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:	CTLA4
NCBI Official Synonym Symbols:	CD; GSE; GRD4; ALPS5; CD152; CTLA-4; IDDM12; CELIAC3
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:	CTLA4
UniProt Entry Name:	CTLA4_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA	Antibodies
Human CTLA4 / CD152 ELISA Kit	Anti-CTLA4 Antibody (CAB13965)
Human CTLA4 (Cytotoxic T Lymphocyte Associated Antigen 4) CLIA Kit (HUES01102)	Anti-CTLA4 Antibody (CAB13966)
	Anti-CTLA4 Antibody (CAB2063)
	CD151 Antibody (PACO07361)
	CHUK Antibody (PACO21234)