人GPX试剂盒说明书-技术文章-上海劲马生物科技有限公司手机版

人GPX试剂盒说明书

时间：2012-04-11 阅读：4338

本试剂盒用于测定人血清、血浆及相关液体样本中GPX）含量。
实验原理
本试剂盒应用双抗体夹心法测定标本中人白介素GPX水平。用纯化的人GPX抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入白介素GPX再与HRP标记的白介素GPX抗体结合，形成抗体-抗原-酶标抗体复合物，经过*洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成zui终的黄色。颜色的深浅和样品中的白介素GPX呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中人白介素GPX）浓度。

如需了解人谷胱甘肽过氧化物酶ELISA试剂盒价格是否有折扣、*或是否有调整,请咨询我司业务人员:,.

Human glutathione peroxidase （GPX）

FOR RESEARCH USE ONLY

Assay range：10ng/L -260ng/L 96 determinations Purpose

This kit allows for the determination of GPX concentrations in Human serum, cell culture supernates and other biological fluids

Principle of the assay

The kit assay Human GPX level in the sample，use Purified Human GPX antibody to coat microtiter plate wells, make solid-phase antibody, then add GPX to wells, Combined GPX antibody which With HRP labeled,become antibody - antigen - enzyme-antibody complex, after washing Compley, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human GPX in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Materials provided with the kit

1	wash solution	20ml× 1bottle	7	Stopp Solution	6ml× 1 bottle
2	HRP-Conjugate reagent	6ml× 1 bottle	8	Standard（480ng/L）	0.5ml× 1 bottle
3	Microelisa stripplate	12well× 8strips	9	Standard diluent	1.5ml× 1bottle
4	Sample diluent	6ml× 1 bottle	10	Instruction	1
5	Chromogen Solution A	6ml× 1 bottle	11	Closure plate membrane	2
6	Chromogen Solution B	6ml× 1 bottle	12	Sealed bags	1

Specimen requirements

1. extract as soon as possible after Specimen collection,and according to the relevant

literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.

2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

Assay procedure

1. Dilute and add sample:Dilute Original density Standard as follow table:

2.add sample：Set blank wells separay （blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same）. testing sample well. add Sample dilution 40μl to testing sample well, then add testing sample 10μl （sample final dilution is 5-fold）, add sample to wells , don’t touch the well wall as far as possible, and Gently mix.

240ng/L	5 Standard	150μl Original density Standard+150μl Standard diluent
120ng/L	4 Standard	150μl 5 Standard+150μl Standard diluent
60ng/L	3 Standard	150μl 4 Standard+150μl Standard diluent
30ng/L	2 Standard	150μl 3 Standard +150μl Standard diluent
15ng/L	1 Standard	150μl 2 Standard +150μl Standard diluent

3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃. 4.Configurate liquid: 30-fold（or 20-fold） wash solution diluted 30-fold （or 20-fold） with distilled water and reserve.

5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.

6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank well.

7.incubate：Operation with 3.

8.washing：Operation with 5.

9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37℃

1 the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue color change to yellow color）.

2 take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and

within 15min.

Steps description

Standard, Sample diluent

Add Standard, Sample diluent, incubate for 30 min at 37℃.

Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.

Wash 5 times,Add Chromogen Solution A and B, incubate for 30 min at 37℃.

Add Stopp Solution

Read absorbance at 450nm within 15 min

calculate

Calculate

Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.

Important notes

1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.

2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.

3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .

4. if the testing material content is excessively higher （The sample OD is bigger than the first

standard well ）,please dilute Sample （n-fold）, Please diluente and multiplied by the dilution factor.（× n× 5）.

5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.

6. The substrate evade the light preservation.

7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.

8. All samples, washing buffer and each kind of reject should according to infective material process.

9. Do not mix reagents with those from other lots.

Storage and validity

1．Storage： 2-8℃.

2．validity： six months