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HUMAN IL-29(IL-29) ELISA Kit

USE INSTRUCTION

FOR RESEARCH USE ONLY

Drug Names

Generic Name：HUMAN IL-29 ELISA Kit.

Purpose

This kit allows for the determination of IL-29 concentrations in human serum, blood plasma, and other biological fluids.

Principle of the assay

The kit use ELISA to assay human IL-29 level in the sample，use Purified human IL-29 antibody to coat microtiter plate wells, make solid-phase antibody, add IL-29 to coated microtiter wells, Combined antibody which With HRP labeled goat anti-human become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution to each well and color reactions, TMB substrate becomes blue color At HRP enzyme-catalyzed, And at the effect of acid the color finally become yellow, the depth of color and the IL-29 of sample were positively correlated, measure the optical densit (OD) at 450 nm with microtiter plate reader, calculate human IL-29 concentration by standard curv.

Materials provided with the kit

Specimen requirements

1.      extract as soon as possible after Specimen collection, Extracted according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can not be tested immediately, 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 to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100μl to the first and the second well, then add Standard dilution 50μl to the first and the second well, mix; take out 100μl form the first and the second well then add it to the third and the forth well separately. then add Standard dilution 50μl to the third and the forth well ,mix ; then take out 50μl from the third and the forth well discard, add 50μl to the fifth and the sixth well ,then add Standard dilution 50μl to the fifth and the sixth well, mix ; take out 50μl from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50μl to the seventh and the eighth well ,mix ; take out 50μl from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50μl to the ninth and the tenth well, mix , take out 50μl from the ninth and the tenth well discard(the Sample volume is 50μl of each well after Diluting,density: (24ng/L,16ng/L , 8ng/L, 4ng/L, 2ng/L)).

2.        add sample：Set blank wells separately (blank comparison wells don’t add sample and ELISA reagent, other each step operation is same). testing sample well. add Sample dilution 40μl to testing sample well which on ELISA plates coated, then add testing sample 10μl (sample final dilute degree is 5 times), add sample to the bottom of ELISA plates coated well , don’t touch the well wall 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: 20 times of Wash Concentrate diluted 20 times with distilled water and reserve.

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

6.        add enzyme：Add ELISA reagents 50μl to each well, except the blank well.

7.        incubate：Operation with 3.

8.         washing：Operation with 5.

9.        color：add color reagent A 50μl and color reagent B 50μl to each well. Gently mix, incubate for 15 min at 37℃.

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

11.    assay：take blank well as zero , measure the optical densit (OD) at 450 nm after Adding Stop Solution and within 15min.

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 multiple, 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  then use, 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.     Please make specification curve when you assay, had better make duplicate well, if in the sample the testing material content is excessively high (The sample OD is bigger than the first standard well OD ),please use Sample dilution to dilute certain multiple (n times),then assay. Please multiply total Dilution Times when calculate（×n×5）.

5.      Closure plate membrane only limits the disposable use, in order to avoid the overlapping pollution.

6.      The substrate please 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.      This reagent which different batch number component do not mix.

10. If it’s different form English instruction, take English instruction as the standard.

Assay range

1.5ng/L -30 ng/L.

Package size 

96 determinations.

Storage and validity

1．Storage： 2-8℃.

2．validity： six months.

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