人間 ECHO-IgG ELISA Kit (HUFI04722)- 高感度

商品名:	Human ECHO-IgG (Enteric Cytopathic Human Orphan virus-Immunoglobulin G) ELISA Kit
製品コード:	HUFI04722
サイズ:	96T
エイリアス:	ECHO-IgG ELISA Kit
検出方法:	Indirect ELISA - Biotin
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Human ECHO-IgG (Enteric Cytopathic Human Orphan virus-Immunoglobulin G) concentrations in serum plasma and other biological fluids.
感度:	Contact Technical Support
範囲:	Contact Technical Support
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 ECHO-IgG (Enteric Cytopathic Human Orphan virus-Immunoglobulin G) and the recovery rates were calculated by comparing the measured value to the expected amount of Human ECHO-IgG (Enteric Cytopathic Human Orphan virus-Immunoglobulin G) in samples. Enquire for more information.
直線性:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ECHO-IgG (Enteric Cytopathic Human Orphan virus-Immunoglobulin G) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
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

*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、37°Cで少なくとも30分間TMB基質を平衡化します。サンプルと試薬を希釈するときは、それらが完全かつ均一に混合されていることを確認してください。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 (diluted at least ½ with Sample Dilution Buffer) and control (blank) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (blank) wells!
2.	Aliquot 100µl of standard solutions into the standard wells.
3.	Add 100µl of properly diluted Sample into the sample wells.
4.	Seal the plate with a cover and incubate at 37 °C for 90 mins.
5.	Wash: Repeat the aspiration/wash process for 2 times.
6.	Add 100µl of Biotin-labelled antibody working solution into the bottom of each well (standard, test sample & zero wells) without touching the side walls.
7.	Seal the plate with a cover and incubate at 37°C for 60 mins.
8.	Wash: Remove the cover, and wash plate 3 times with Wash buffer.
9.	HRP-Streptavidin Conjugate (SABC): Add 100?l of SABC Working Solution into each well, cover the plate and incubate at 37°C for 30 minutes.
10.	Wash: Remove the cover and wash plate 5 times with Wash Buffer.
11.	TMB Substrate: Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark for 10-20 mins. (Note: This incubation time is for reference only, the optimal time should be determined by the end-user.) As soon as a blue colour develops in the first 3-4 wells (with most concentrated standards) and the other wells show no obvious colour, terminate the reaction.
12.	Add 50µl of Stop solution into each well and mix thoroughly. The colour changes into yellow immediately.
13.	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.