Human Thyrotropin receptor (TSHR) ELISA Kit

Product Name:	Human Thyrotropin receptor (TSHR) ELISA Kit
SKU:	HUEB2712
Size:	96T
Target:	Human Thyrotropin receptor (TSHR)
Synonyms:	Thyroid-stimulating hormone receptor, TSH-R, LGR3
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	31.2-2000pg/mL
Sensitivity:	15.3pg/mL

Intra CV:

4.9%

Inter CV:

8.5%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	100-109%	106-116%	94-106%	96-107%
EDTA Plasma(N=5)	90-100%	103-115%	105-115%	89-100%
Heparin Plasma(N=5)	108-117%	110-120%	97-106%	101-111%

Recovery:

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

Function:

Receptor for the thyroid-stimulating hormone (TSH) or thyrotropin (PubMed:11847099, PubMed:12045258). Also acts as a receptor for the heterodimeric glycoprotein hormone (GPHA2:GPHB5) or thyrostimulin (PubMed:12045258). The activity of this receptor is mediated by G proteins which activate adenylate cyclase (PubMed:11847099). Plays a central role in controlling thyroid cell metabolism.

Uniprot:	P16473
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Thyrotropin receptor
Sub Unit:	Interacts with heterodimer GPHA2:GPHB5; this interaction stimulates cAMP production (PubMed:12045258). Interacts (via the PDZ-binding motif) with SCRIB; regulates TSHR trafficking and function (PubMed:15775968).
Subcellular Location:	Cell membrane Multi-pass membrane protein Basolateral cell membrane Multi-pass membrane protein
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	TSHR: Receptor for thyrothropin. Plays a central role in controlling thyroid cell metabolism. The activity of this receptor is mediated by G proteins which activate adenylate cyclase. Also acts as a receptor for thyrostimulin (GPA2+GPB5). Defects in TSHR are found in patients affected by hyperthyroidism with different etiologies. Somatic, constitutively activating TSHR mutations and/or constitutively activating G(s)alpha mutations have been identified in toxic thyroid nodules (TTNs) that are the predominant cause of hyperthyroidism in iodine deficient areas. These mutations lead to TSH independent activation of the cAMP cascade resulting in thyroid growth and hormone production. TSHR mutations are found in autonomously functioning thyroid nodules (AFTN), toxic multinodular goiter (TMNG) and hyperfunctioning thyroid adenomas (HTA). TMNG encompasses a spectrum of different clinical entities, ranging from a single hyperfunctioning nodule within an enlarged thyroid, to multiple hyperfunctioning areas scattered throughout the gland. HTA are discrete encapsulated neoplasms characterized by TSH- independent autonomous growth, hypersecretion of thyroid hormones, and TSH suppression. Defects in TSHR are also a cause of thyroid neoplasms (papillary and follicular cancers). Autoantibodies against TSHR are directly responsible for the pathogenesis and hyperthyroidism of Graves disease. Antibody interaction with TSHR results in an uncontrolled receptor stimulation. Defects in TSHR are the cause of congenital hypothyroidism non-goitrous type 1 (CHNG1); also known as congenital hypothyroidism due to TSH resistance. CHNG1 is a non-autoimmune condition characterized by resistance to thyroid- stimulating hormone (TSH) leading to increased levels of plasma TSH and low levels of thyroid hormone. CHNG1 presents variable severity depending on the completeness of the defect. Most patients are euthyroid and asymptomatic, with a normal sized thyroid gland. Only a subset of patients develop hypothyroidism and present a hypoplastic thyroid gland. Defects in TSHR are the cause of familial gestational hyperthyroidism (HTFG). HTFG is a condition characterized by abnormally high levels of serum thyroid hormones occurring during early pregnancy. Defects in TSHR are the cause of hyperthyroidism non- autoimmune (HTNA). It is a condition characterized by abnormally high levels of serum thyroid hormones, thyroid hyperplasia, goiter and lack of anti-thyroid antibodies. Typical features of Graves disease such as exophthalmia, myxedema, antibodies anti-TSH receptor and lymphocytic infiltration of the thyroid gland are absent. Belongs to the G-protein coupled receptor 1 family. FSH/LSH/TSH subfamily. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:GPCR, family 1; Receptor, GPCR; Membrane protein, multi-pass; Membrane protein, integral Chromosomal Location of Human Ortholog: 14q31 Cellular Component: integral to plasma membrane; plasma membrane; receptor complex Molecular Function:protein binding; thyroid-stimulating hormone receptor activity Biological Process: G-protein coupled receptor protein signaling pathway; G-protein signaling, coupled to cyclic nucleotide second messenger; cell-cell signaling; positive regulation of cell proliferation Disease: Hyperthyroidism, Nonautoimmune; Hypothyroidism, Congenital, Nongoitrous, 1; Hyperthyroidism, Familial Gestational
NCBI Summary:	The protein encoded by this gene is a membrane protein and a major controller of thyroid cell metabolism. The encoded protein is a receptor for thyrothropin and thyrostimulin, and its activity is mediated by adenylate cyclase. Defects in this gene are a cause of several types of hyperthyroidism. Three transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Dec 2008]
UniProt Code:	P16473
NCBI GenInfo Identifier:	62298994
NCBI Gene ID:	7253
NCBI Accession:	P16473.2
UniProt Secondary Accession:	P16473,Q16503, Q8TB90, Q96GT6, Q9P1V4, Q9ULA3, Q9UPH3 A0PJU7, F5GYU5, G3V2A9,
UniProt Related Accession:	P16473
Molecular Weight:	86,830 Da
NCBI Full Name:	Thyrotropin receptor
NCBI Synonym Full Names:	thyroid stimulating hormone receptor
NCBI Official Symbol:	TSHR
NCBI Official Synonym Symbols:	LGR3; CHNG1; hTSHR-I
NCBI Protein Information:	thyrotropin receptor; thyrotropin receptor-I, hTSHR-I; seven transmembrane helix receptor; thyroid stimulating hormone receptor, isoform 2
UniProt Protein Name:	Thyrotropin receptor
UniProt Synonym Protein Names:	Thyroid-stimulating hormone receptor
UniProt Gene Name:	TSHR
UniProt Entry Name:	TSHR_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

Human TSHR / Thyrotropin receptor ELISA Kit