人間 HAAO ELISA Kit (HUFI00965)- 高感度

商品名:	Human HAAO / 3-hydroxyanthranilate 3,4-dioxygenase ELISA Kit
製品コード:	HUFI00965
サイズ:	96T
エイリアス:	HAAO, 3-hydroxyanthranilate 3, 4-dioxygenase, 3-HAO, HAD, 3-hydroxyanthranilate oxygenase, 3-hydroxyanthranilic acid dioxygenase
検出方法:	Sandwich ELISA, Double Antibody
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Human HAAO concentrations in serum plasma and other biological fluids.
感度:	0.188ng/ml
範囲:	0.313-20ng/ml
保管所:	4°C for 6 months
ノート:	For Research Use Only

回復:

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.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-98	94
EDTA plasma(n=5)	85-101	93
UFH plasma(n=5)	87-104	97

直線性:

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.

Sample	1:2	1:4	1:8
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%

成分	量	保管所
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

必要なその他の材料と設備:

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

Uniprot

P46952

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

*ノート: プロトコルは、各バッチ/ロットに固有です。正しい手順については、キットに含まれているプロトコルに従ってください。

ウェルに加える前に、SABCワーキング溶液とTMB基質を37°Cで少なくとも30分間平衡化します。サンプルと試薬を希釈するときは、完全に均一に混合する必要があります。各テストの標準曲線をプロットすることをお勧めします。

ステップ	プロトコル
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.

ELISAアッセイを実施する場合、可能な限り最良の結果を達成するためにサンプルを準備することが重要です。以下に、さまざまなサンプルタイプのサンプルを準備するための手順のリストを示します。

サンプルタイプ	プロトコル
血清	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.
プラズマ	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.
尿および脳脊髄液	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.
細胞培養上清	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.
細胞溶解物	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.
組織ホモジネート	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.
組織溶解物	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.
母乳	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.