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Human HAAO / 3-hydroxyanthranilate 3,4-dioxygenase ELISA Kit

SKU:
HUFI00965
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P46952
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich
Synonyms:
HAAO, 3-hydroxyanthranilate 3, 4-dioxygenase, 3-HAO, HAD, 3-hydroxyanthranilate oxygenase, 3-hydroxyanthranilic acid dioxygenase
Reactivity:
Human
Research Area:
Metabolism
€599
Frequently bought together:

Description

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Human HAAO/3-hydroxyanthranilate 3,4-dioxygenase ELISA Kit

The Human HAAO (3-Hydroxyanthranilate 3,4-dioxygenase) ELISA Kit is a powerful tool for the precise measurement of HAAO levels in human samples, including serum, plasma, and cell culture supernatants. With its exceptional sensitivity and specificity, this kit delivers accurate and consistent results, making it a valuable asset for a wide array of research endeavors.HAAO is an important enzyme involved in the kynurenine pathway, playing a crucial role in the metabolism of tryptophan. Dysregulation of HAAO has been implicated in various disorders, including psychiatric illnesses, autoimmune diseases, and neurodegenerative conditions.

By accurately quantifying HAAO levels, researchers can gain insights into the underlying mechanisms of these disorders and potentially identify new therapeutic targets.Overall, the Human HAAO ELISA Kit is an indispensable tool for researchers looking to investigate the role of HAAO in health and disease, offering precise and reliable measurements for impactful research outcomes.

Product Name:Human HAAO / 3-hydroxyanthranilate 3,4-dioxygenase ELISA Kit
Product Code:HUFI00965
Size:96 Assays
Alias:HAAO, 3-hydroxyanthranilate 3, 4-dioxygenase, 3-HAO, HAD, 3-hydroxyanthranilate oxygenase, 3-hydroxyanthranilic acid dioxygenase
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human HAAO concentrations in serum plasma and other biological fluids.
Sensitivity:0.188ng/ml
Range:0.313-20ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human HAAO and the recovery rates were calculated by comparing the measured value to the expected amount of Human HAAO in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)85-9894
EDTA plasma(n=5)85-10193
UFH plasma(n=5)87-10497
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human HAAO and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)89-103%86-98%92-101%
EDTA plasma(n=5)83-97%86-95%84-100%
UFH plasma(n=5)83-92%87-100%81-94%
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
UniprotP46952
UniProt Protein Function:HAAO: Catalyzes the oxidative ring opening of 3- hydroxyanthranilate to 2-amino-3-carboxymuconate semialdehyde, which spontaneously cyclizes to quinolinate. Belongs to the 3-HAO family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:Protein type:Oxidoreductase; Amino Acid Metabolism - tryptophan; EC 1.13.11.6

Chromosomal Location of Human Ortholog: 2p21

Cellular Component: mitochondrial membrane; cytosol

Molecular Function:electron carrier activity; ferrous iron binding; oxygen binding; 3-hydroxyanthranilate 3,4-dioxygenase activity

Biological Process: anthranilate metabolic process; response to cadmium ion; response to zinc ion; quinolinate biosynthetic process; tryptophan catabolic process

NCBI Summary:3-Hydroxyanthranilate 3,4-dioxygenase is a monomeric cytosolic protein belonging to the family of intramolecular dioxygenases containing nonheme ferrous iron. It is widely distributed in peripheral organs, such as liver and kidney, and is also present in low amounts in the central nervous system. HAAO catalyzes the synthesis of quinolinic acid (QUIN) from 3-hydroxyanthranilic acid. QUIN is an excitotoxin whose toxicity is mediated by its ability to activate glutamate N-methyl-D-aspartate receptors. Increased cerebral levels of QUIN may participate in the pathogenesis of neurologic and inflammatory disorders. HAAO has been suggested to play a role in disorders associated with altered tissue levels of QUIN. [provided by RefSeq, Jul 2008]
UniProt Code:P46952
NCBI GenInfo Identifier:308153402
NCBI Gene ID:23498
NCBI Accession:P46952.2
UniProt Secondary Accession:P46952,Q53QZ7, Q8N6N9, A6NE56, B4DIN2,
UniProt Related Accession:P46952
Molecular Weight:17,275 Da
NCBI Full Name:3-hydroxyanthranilate 3,4-dioxygenase
NCBI Synonym Full Names:3-hydroxyanthranilate 3,4-dioxygenase
NCBI Official Symbol:HAAO
NCBI Official Synonym Symbols:HAO; 3-HAO
NCBI Protein Information:3-hydroxyanthranilate 3,4-dioxygenase; HAD; 3-hydroxyanthranilate oxygenase; 3-hydroxyanthranilic acid dioxygenase
UniProt Protein Name:3-hydroxyanthranilate 3,4-dioxygenase
UniProt Synonym Protein Names:3-hydroxyanthranilate oxygenase; 3-HAO; 3-hydroxyanthranilic acid dioxygenase; HAD
UniProt Gene Name:HAAO
UniProt Entry Name:3HAO_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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