Human TNF-α Elisa Test Kit

INTENDED USE

This kit a lows for the determination of TNF-α concentrations in Human ce l culture supernates.

Assay procedure

1. Dilute and add sample:Dilute Original density Standard as fo low table: 400ng/L 5 Standard 150μl Original density Standard+150μl Standard diluent 200ng/L 4 Standard 150μl 5 Standard+150μl Standard diluent 100ng/L 3 Standard 150μl 4 Standard+150μl Standard diluent 50ng/L 2 Standard 150μl 3 Standard +150μl Standard diluent 25ng/L 1 Standard 150μl 2 Standard +150μl Standard diluent

2.add sample:Set blank wels separately (blank comparison wels don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample we l. Add 50μl of standard to Microelisa stripplate,add Sample dilution 40μl to testing sample we l, then add testing sample 10μl (sample final dilution is 5-fold), add sample to we ls , don’t touch the we l wal as far as possible, and Gently mix.

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 disti led water and reserve.

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

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

7.incubate:Operation with 3.

8.washing:Operation with 5.

9.color:Add Chromogen Solution A 50ul and Chromogen Solution B 50ul to each wel, evade the light preservation for 10 min at 37℃

10.Stop the reaction:Add Stop Solution50μl to each wel, Stop the reaction(the blue color change to yelow color). 

11.assay:take blank we l as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.

Specimen requirements

1. extract as soon as possible after Specimen colection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, 1 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.

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 wil Crystalization 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 Voley .

4. if the testing material content is excessively higher (The sample OD is bigger than the first standard we l ),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. Thesubstrate 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. Al samples, washing buffer and each kind of reject should according to infective material process.

9. Donotmix reagents with those from other lots.