Human Inosine triphosphate pyrophosphatase / ITPA ELISA Kit

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SKU:
HUFI01327
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9BY32
Sensitivity:
0.469ng/ml
Range:
0.781-50ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
ITPA, Inosine triphosphate pyrophosphatase, ITPase, Inosine triphosphatase, Putative oncogene protein hlc14-06-p, Nucleoside-triphosphate pyrophosphatase, NTPase, Nucleoside-triphosphate diphosphatase, Non-canonical purine NTP pyrophosphatase, Non-st
Reactivity:
Human
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Description

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Product Name:Human Inosine triphosphate pyrophosphatase / ITPA ELISA Kit
Product Code:HUFI01327
Size:96 Assays
Alias:ITPA, Inosine triphosphate pyrophosphatase, ITPase, Inosine triphosphatase, Putative oncogene protein hlc14-06-p, Nucleoside-triphosphate pyrophosphatase, NTPase, Nucleoside-triphosphate diphosphatase, Non-canonical purine NTP pyrophosphatase, Non-standard purine NTP pyrophosphatase, My049, OK, SW-cl.9, C20orf37
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human ITPA concentrations in serum plasma and other biological fluids.
Sensitivity:0.469ng/ml
Range:0.781-50ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human ITPA and the recovery rates were calculated by comparing the measured value to the expected amount of Human ITPA in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)91-10598
EDTA plasma(n=5)85-10493
UFH plasma(n=5)88-10595
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ITPA and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)90-102%86-104%85-97%
EDTA plasma(n=5)82-98%97-100%83-101%
UFH plasma(n=5)83-100%84-100%83-100%
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
UniprotQ9BY32
UniProt Protein Function:ITPA: Pyrophosphatase that hydrolyzes the non-canonical purine nucleotides inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) as well as 2'-deoxy-N-6-hydroxylaminopurine triposphate (dHAPTP) and xanthosine 5'-triphosphate (XTP) to their respective monophosphate derivatives. The enzyme does not distinguish between the deoxy- and ribose forms. Probably excludes non-canonical purines from RNA and DNA precursor pools, thus preventing their incorporation into RNA and DNA and avoiding chromosomal lesions. Defects in ITPA are the cause of inosine triphosphate pyrophosphohydrolase deficiency (ITPAD). It is a common inherited trait characterized by the abnormal accumulation of inosine triphosphate (ITP) in erythrocytes and also leukocytes and fibroblasts. The pathological consequences of ITPA deficiency, if any, are unknown. However, it might have pharmacogenomic implications and be related to increased drug toxicity of purine analog drugs. Three different human populations have been reported with respect to their ITPase activity: high, mean (25% of high) and low activity. The variant Thr-32 is associated with complete loss of enzyme activity, may be by altering the local secondary structure of the protein. Heterozygotes for this polymorphism have 22.5% of the control activity: this is consistent with a dimeric structure of the enzyme. Belongs to the HAM1 NTPase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:Protein type:Xenobiotic Metabolism - drug metabolism - other enzymes; Nucleotide Metabolism - pyrimidine; EC 3.6.1.19; Hydrolase; Nucleotide Metabolism - purine

Chromosomal Location of Human Ortholog: 20p

Cellular Component: cytoplasm; cytosol

Molecular Function:metal ion binding; nucleotide binding

Biological Process: deoxyribonucleoside triphosphate catabolic process; nucleobase, nucleoside and nucleotide metabolic process; ITP catabolic process; chromosome organization and biogenesis

Disease: Inosine Triphosphatase Deficiency

NCBI Summary:This gene encodes an inosine triphosphate pyrophosphohydrolase. The encoded protein hydrolyzes inosine triphosphate and deoxyinosine triphosphate to the monophosphate nucleotide and diphosphate. This protein, which is a member of the HAM1 NTPase protein family, is found in the cytoplasm and acts as a homodimer. Defects in the encoded protein can result in inosine triphosphate pyrophosphorylase deficiency which causes an accumulation of ITP in red blood cells. Alternate splicing results in multiple transcript variants. [provided by RefSeq, Jun 2012]
UniProt Code:Q9BY32
NCBI GenInfo Identifier:30173120
NCBI Gene ID:3704
NCBI Accession:Q9BY32.2
UniProt Secondary Accession:Q9BY32,O14878, Q5JWH4, Q9BYN1, Q9BYX0, Q9H3H8, A2A2N2 A4UIM5, B2BCH7,
UniProt Related Accession:Q9BY32
Molecular Weight:21,446 Da
NCBI Full Name:Inosine triphosphate pyrophosphatase
NCBI Synonym Full Names:inosine triphosphatase (nucleoside triphosphate pyrophosphatase)
NCBI Official Symbol:ITPA
NCBI Official Synonym Symbols:My049; C20orf37; dJ794I6.3; HLC14-06-P
NCBI Protein Information:inosine triphosphate pyrophosphatase; ITPase; NTPase; inosine triphosphatase-A; putative oncogene protein HLC14-06-P; nucleoside-triphosphate diphosphatase; non-standard purine NTP pyrophosphatase; non-canonical purine NTP pyrophosphatase; inosine triphosphate pyrophosphohydrolase
UniProt Protein Name:Inosine triphosphate pyrophosphatase
UniProt Synonym Protein Names:Non-canonical purine NTP pyrophosphatase; Non-standard purine NTP pyrophosphatase; Nucleoside-triphosphate diphosphatase; Nucleoside-triphosphate pyrophosphatase; NTPase; Putative oncogene protein hlc14-06-p
UniProt Gene Name:ITPA
UniProt Entry Name:ITPA_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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