VA__Vitamin_A__ELISA_Kit_.pdf
《VA__Vitamin_A__ELISA_Kit_.pdf》由会员分享,可在线阅读,更多相关《VA__Vitamin_A__ELISA_Kit_.pdf(11页珍藏版)》请在得力文库 - 分享文档赚钱的网站上搜索。
1、TEL:021-60514606Web:www.sh-Catalog#:yb-0006c VA(Vitamin A)ELISA Kit VA(Vitamin A)ELISA Kit Product Manual Catalog No:YB-0006c (FOR RESEARCH USE ONLY.DO NOT USE IT IN CLINICAL DIAGONOSIS!)Dear customer,Thank you for choosing our products.Please read the instructions carefully before use and check all
2、 the reagent compositions!If in doubt,please contact Elabscience.Kit Components:Item Specifications Storage Micro ELISA Plate(Dismountable)812 or 86*4C/-20#Reference Standard 2/1vial*4C/-20#Reference Standard&Sample Diluent 1vial 20mL/12mL*4C Concentrated Biotinylated Detection Ab 1vial 120L/70L*4C/
3、-20#Biotinylated Detection Ab Diluent 1vial 10mL/6mL*4C Concentrated HRP Conjugate 1vial 120L/70L*4C(shading light)HRP Conjugate Diluent 1vial 10mL/6mL*4C Concentrated Wash Buffer(25)1vial 30mL/16mL*4C Substrate Reagent 1vial 10mL/6mL*4C(shading light)Stop Solution 1vial 10mL/6mL*4C Plate Sealer 5/3
4、pieces*Product Description 1 copy Certificate of Analysis 1 copy Note:*:96T/48T#:Its OK to keep the kit in 4,if the kit is scheduled to be used up in one week.Please keep the reagent in-20 for long-term storage or repeated use.The reagent in each vial is slightly more that its volume written on labe
5、l,please take out the required volume by certain tools(such as transferpettor,measuring cylinder),rather than pouring directly.Test principle This ELISA kit uses Competitive-ELISA as the method.The microtiter plate provided in this kit has been pre-coated with VA.During the reaction,VA in the sample
6、 or standard competes with a fixed amount of VA on the solid phase supporter for sites on the Biotinylated Detection Ab specific to VA.Excess conjugate and unbound sample or standard are washed from the plate,and TEL:021-60514606Web:www.sh-Catalog#:yb-0006c Avidin conjugated to Horseradish Peroxidas
7、e(HRP)is added to each microplate well and incubated.Then a TMB substrate solution is added to each well.The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm 2 nm.The concentration o
8、f VA in the samples is then determined by comparing the O.D.of the samples to the standard curve.6th Edition,revised in June,2014 9 Sample collection and storage Serum-Allow samples to clot for 2 hours at room temperature or overnight at 4C before centrifugation for 20 minutes at approximately 1000g
9、.Collect the supernatant and carry out the assay immediately.Blood collection tubes should be disposable,non-pyrogenic,and non-endotoxin.Plasma-Collect plasma using EDTA-Na2 or heparin as an anticoagulant.Centrifuge samples for 15 minutes at 1000g at 2-8C within 30 minutes of collection.Collect the
10、supernatant and carry out the assay immediately.Avoid hemolysis,high cholesterol samples.Tissue homogenates For general information,hemolysis blood may affect the result,so you should rinse the tissues with ice-cold PBS(0.01M,pH=7.4)to remove excess blood thoroughly.Tissue pieces should be weighed a
11、nd then minced to small pieces which will be homogenized in PBS(the volume depends on the weight of the tissue.9mL PBS would be appropriate to 1 gram tissue pieces.Some protease inhibitor is recommended to add into the PBS.)with a glass homogenizer on ice.To further break the cells,you can sonicate
12、the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles.The homogenates are then centrifugated for 5minutes at 5000g to get the TEL:021-60514606Web:www.sh-Catalog#:yb-0006c supernate.Cell culture supernate Centrifuge supernate for 20 minutes to remove insoluble impurity
13、and cell debris at 1000g at 2-8C.Collect the clear supernate and carry out the assay immediately.Other biological fluids Centrifuge samples for 20 minutes at 1000g at 2-8C.Collect the supernatant and carry out the assay immediately.(You can refer to our website for detailed processing method:http:/
14、preparation Samples should be clear and transparent and be centrifuged to remove suspended solids.Note:Serum and plasma to be used within 7 days when stored at 2-8C,otherwise samples must be divided and stored at-20C(1 month)or-80C(6 months)to avoid loss of bioactivity and contamination.Avoid freeze
15、-thaw cycles.When performing the assay slowly bring samples to room temperature.If the sample concentration is higher than the maximum standard value,please dilute it with appropriate factor according to the actual situation.(A pre-test is recommended to determine the dilute factor).Other supplies r
16、equired Microplate reader with 450nm wavelength filter High-precision transferpettor,EP tubes and disposable pipette tips 37C Incubator,Deionized or distilled water Absorbent paper 6th Edition,revised in June,2014 10 Reagent preparation Bring all reagents to room temperature before use.Wash Buffer-D
17、ilute 30mL of Concentrated Wash Buffer into 750 mL of Wash Buffer with deionized or distilled water.Put unused solution back at 4C.If crystals have formed in the concentrate,you can TEL:021-60514606Web:www.sh-Catalog#:yb-0006c warm it with 40C water bath(Heating temperature should not exceed 50C)and
18、 mix it gently until the crystals have completely dissolved.The solution should be cooled to room temperature before use.Standard-Centrifuge at 10,000g for 1 minute,and reconstitute the Standard with 1.0mL of Reference Standard&Sample Diluent.Tighten the lid,let it stand for 10minutes and turn it up
19、side down for several times.After it dissolves fully,mix it thoroughly with a pipette.This reconstitution produces a stock solution of 10ng/mL.Then make serial dilutions as needed(Making serial dilution in the wells directly is not permitted).The recommended concentrations are as follows:10、5、2.5、1.
20、25、0.625、0.313、0.156、0ng/mL.As if you want to make standard solution at the concentration of 5ng/mL,you can take 0.5mL the standard at 10ng/mL,add it to an EP tube with 0.5mL Reference Standard&Sample Diluent,and mix it.The procedures of making the remaining concentrations are all the same.The undil
21、uted standard serves as the highest standard(10ng/mL).The Reference Standard&Sample Diluent serves as the zero(0ng/mL).(500L/tube,for example.Can also be diluted according to the actual amount,such as 200L/tube)1 2 3 4 5 6 7 8 Tube 10 5 2.5 1.25 0.625 0.313 0.156 0 ng/mL Biotinylated Detection Ab Ca
22、lculate the required amount before experiment(50L/well).In actual preparation you should prepare 100200 L more.Dilute the concentrated Biotinylated Detection Ab to the working concentration using Biotinylated Detection Ab Diluent(1:100).Concentrated HRP Conjugate Calculate the required amount before
23、 experiment(100L/well).In actual preparation you should prepare 100200L more.Dilute the Concentrated HRP Conjugate to the working concentration using Concentrated HRP Conjugate Diluent(1:100).6th Edition,revised in June,2014 TEL:021-60514606Web:www.sh-Catalog#:yb-0006c 11 Washing Procedure:1.Automat
24、ed washer:add 350L wash buffer into each well,the interval between injection and suction should be set about 60s.2.Manual wash:add 350L wash buffer into each well,soak it for 12minutes,suck(no inside wall touching)or get rid of liquid within the micro ELISA plate and pat it dry on thick clean absorb
25、ent paper.Assay procedure Allow all reagents to reach room temperature All the reagents should be mixed thoroughly by gently swirling before pipetting.Avoid foaming.1.Add Sample and Biotinylated Detection Ab:Add 50L of Standard,Blank,or Sample per well.The blank well is added with Reference Standard
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- VA_Vitamin_A_ELISA_Kit_
限制150内