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Human Thiopurine S-methyltransferase / TMPT ELISA Kit

SKU:
HUFI00917
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P51580
Sensitivity:
1.875ng/ml
Range:
3.125-200ng/ml
ELISA Type:
Sandwich
Synonyms:
TPMT, Thiopurine S-methyltransferase, Thiopurine methyltransferase
Reactivity:
Human
Research Area:
Cell Biology
€599
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Description

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Human Thiopurine S-methyltransferase / TMPT ELISA Kit

Thiopurine drugs are metabolized by TMPT. Thiopurine drugs, such as 6-mercaptopurine, are utilized in chemotherapy. Genetic polymorphisms that modify this enzymatic activity are connected with differences in sensitivity and toxicity to such medicines among people, resulting in thiopurine S-methyltransferase insufficiency. On chromosomes 3, 18 and X, three different types of pseudogenes have been discovered. The Assay Genie Thiopurine S-methyltransferase / TMPT ELISA is a highly sensitive assay for the quantitative measurement of Thiopurine S-methyltransferase / TMPT in serum, blood, plasma, cell culture supernatant and tissue samples.

Product Name:Human Thiopurine S-methyltransferase / TMPT ELISA Kit
Product Code:HUFI00917
Size:96 Assays
Alias:TPMT, Thiopurine S-methyltransferase, Thiopurine methyltransferase
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human TPMT concentrations in serum plasma and other biological fluids.
Sensitivity:1.875ng/ml
Range:3.125-200ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human TPMT and the recovery rates were calculated by comparing the measured value to the expected amount of Human TPMT in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)92-10498
EDTA plasma(n=5)89-10297
UFH plasma(n=5)88-9895
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TPMT and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-97%92-105%88-105%
EDTA plasma(n=5)82-96%83-100%85-98%
UFH plasma(n=5)82-99%90-100%84-99%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

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
UniprotP51580
UniProt Protein Function:TPMT: Catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. Defects in TPMT are the cause of thiopurine S- methyltransferase deficiency (TPMT deficiency). TPMT is an enzyme involved in the normal metabolic inactivation of thiopurine drugs. These drugs are generally used as immunosupressants or cytotoxic drugs and are prescribed for a variety of clinical conditions including leukemia, autoimmune disease and organ transplantation. Patients with intermediate or no TPMT activity are at risk of toxicity after receiving standard doses of thiopurine drugs and it is shown that inter-individual differences in response to these drugs are largely determined by genetic variation at the TPMT locus. Belongs to the methyltransferase superfamily. TPMT family.
UniProt Protein Details:Protein type:Xenobiotic Metabolism - drug metabolism - other enzymes; EC 2.1.1.67; Methyltransferase

Chromosomal Location of Human Ortholog: 6p22.3

Cellular Component: cytosol

Molecular Function:thiopurine S-methyltransferase activity

Biological Process: methylation; xenobiotic metabolic process; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

Disease: Thiopurine S-methyltransferase Deficiency

NCBI Summary:This gene encodes the enzyme that metabolizes thiopurine drugs via S-adenosyl-L-methionine as the S-methyl donor and S-adenosyl-L-homocysteine as a byproduct. Thiopurine drugs such as 6-mercaptopurine are used as chemotherapeutic agents. Genetic polymorphisms that affect this enzymatic activity are correlated with variations in sensitivity and toxicity to such drugs within individuals, causing thiopurine S-methyltransferase deficiency. Related pseudogenes have been identified on chromosomes 3, 18 and X. [provided by RefSeq, Aug 2014]
UniProt Code:P51580
NCBI GenInfo Identifier:1730006
NCBI Gene ID:7172
NCBI Accession:P51580.1
UniProt Secondary Accession:P51580,O14806, O15423, O15424, O15425, O15426, O15515 O15548, O43213, Q5VUK6, Q9UBE6, Q9UBT8,
UniProt Related Accession:P51580
Molecular Weight:245
NCBI Full Name:Thiopurine S-methyltransferase
NCBI Synonym Full Names:thiopurine S-methyltransferase
NCBI Official Symbol:TPMT
NCBI Protein Information:thiopurine S-methyltransferase; S-adenosyl-L-methionine:thiopurine S-methyltransferase
UniProt Protein Name:Thiopurine S-methyltransferase
UniProt Synonym Protein Names:Thiopurine methyltransferase
UniProt Gene Name:TPMT
UniProt Entry Name:TPMT_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.

StepProtocol
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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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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