Human interleukin 6 (IL-6) enzyme-linked immunosorbent assay (ELISA) kit instruction manual This reagent is for research use only Purpose: This kit is used to determine the content of interleukin-6 (IL-6) in human serum, plasma and related fluid samples. Experimental principle: Kit composition: Sample processing and requirements: Steps Precautions: Calculation: Kit performance: Blowing Mouth,Disposable Blowing Mouth,Filter Spirometer Mouthpiece,Disposable Spirometer Mouthpiece Hengshui Qifei Paper Products Co. LTD , https://www.hengshuiqifei.com
This kit uses the double antibody sandwich method to determine the level of human interleukin-6 (IL-6) in the specimen. The microplate was coated with purified human interleukin-6 (IL-6) antibody to prepare a solid phase antibody, and the interleukin was sequentially added to the microwell of the coated monoclonal antibody.
-6 (IL-6), which binds to HRP-labeled interleukin-6 (IL-6) antibody to form an antibody-antigen-enzyme-labeled antibody complex, which is thoroughly washed and then plated with TMB. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with interleukin-6 (IL-6) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the human interleukin-6 (IL-6) in the sample was calculated from the standard curve.
content.
Kit Composition 48-well configuration 96-well configuration Storage instructions 1 copy 1 part sealing film 2 pieces (48) 2 pieces (96)
Sealed bag 1 piece of enzyme label package 1×48 1×96 2-8°C Preservation standard: 9ng/L 0.5ml×1 bottle 0.5ml×1 bottle 2-8°C preservation standard dilution 1.5ml× 1 bottle 1.5ml × 1 bottle 2-8 ° C preservation enzyme standard reagent 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C preservation sample dilution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C preservation color Agent A solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation developer B solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation solution 3 ml × 1 bottle 6 ml × 1 bottle 2 Store concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle at 8 ° C 2-8 ° C
1. Serum: The blood is naturally coagulated for 10-20 minutes at room temperature and centrifuged for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again.
2. Plasma: EDTA or sodium citrate should be selected as anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge.
About 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again.
3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to.
4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm /
Minute). Collect the supernatant carefully. When the intracellular components were detected, the cell suspension was diluted with PBS (pH 7.2-7.4) to a cell concentration of about 1 million/ml. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifugation 20 points
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Around the clock (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
5. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use.
6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided.
7. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
1. Dilution and loading of standard products: 10 wells of standard wells on the enzyme label coating plate, 100 μl of standard in the first and second holes, and then add standard in the first and second holes. 50 μl of the dilution, mix; then add 100 μl from each of the first and second wells to the third and fourth wells, and then add 50 μl of the standard dilution to the third and fourth wells, respectively.
Mix well; then discard 50μl in each of the third and fourth wells, then add 50μl to each of the fifth and sixth wells, and then add 50ul of standard dilution in the fifth and sixth wells. Mix well; after mixing, take 50μl from each of the fifth and sixth holes and add them to the seventh and eighth holes respectively, then add 50μl of the standard dilution solution in the seventh and eighth holes respectively, and mix them from the first 7. Add 50 μl to the ninth and tenth holes in the eighth well, and then add 50 μl of the standard dilution to the ninth and tenth holes. After mixing, take 50 μl from each of the ninth and tenth holes and discard. (The amount of each well after dilution is 50μl,
The concentrations were 6 ng/L, 4 ng/L, 2 ng/L, 1 ng/L, 0.5 ng/L, respectively.
2. Adding samples: Set blank holes separately (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Add 40 μl of the sample dilution to the sample well to be tested on the enzyme-labeled plate, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.
3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
4. Dosing: Dilute 30 (20 times of 48T) concentrated washing solution with distilled water 30 (20 times of 48T) and set aside.
5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Wash: Operate the same as 5.
9. Color development: add 50 μl of color developer A50 to each well, then add 50 μl of color developer B, gently shake and mix, and avoid coloration at 37 °C.
15 minutes.
10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.
1. The kit should be taken out from the refrigerated environment and allowed to equilibrate for 15-30 minutes at room temperature. If the enzyme label is unsealed after unsealing, the strip should be stored in a sealed bag.
2. Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.
3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple (×n×5).
5. The sealing film is intended for single use only to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading.
8. All samples, washings and various wastes should be treated as infectious materials.
9. The different batch components of this reagent must not be mixed.
10. If it is different from the English manual, the English manual shall prevail.
The concentration of the standard is the abscissa and the OD is the ordinate.
Draw a standard curve on the coordinate paper, depending on the OD of the sample
The value is determined from the standard curve by the corresponding concentration; multiplied by the dilution factor; or the linear regression equation of the standard curve is calculated from the concentration of the standard and the OD value, and the OD value of the sample is substituted into the equation to calculate the sample concentration, and then multiplied by The dilution factor is the actual concentration of the sample.
(This picture is for reference only)
1. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value is 0.95 or more.
2. Within and within the batch should be less than 9% and 11% respectively
examination range:
0.4ng/L -9ng/L
Storage conditions and expiration date:
1. Kit storage: 2-8 ° C.
2. Validity: 6 months
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Human Interleukin 6
FOR RESEARCH USE ONLY
Drug Names
Generic Name: Human Interleukin 6 (IL-6) ELISA Kit.
Purpose
This kit allows for the determination of IL-6 concentrations in Human serum.
Principle of the assay
The kit assay Human IL-6 level in the sample, use Purified Human IL-6 antibody to coat
Microtiter plate wells, make solid-phase antibody, then add IL-6 to wells, Combined IL-6
Antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after
Washing Completely, 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 IL-6 in the samples is then determined by comparing the OD of the samples
To the standard curve.
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Materials provided with the kit
Materials provided with
The kit
48determinations 96 determinations Storage
User manual 1 1
Closure plate membrane 2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8°C
Standard: 9ng/L 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8°C
Standard diluent 1.5ml×1 bottle 1.5ml×1 bottle 2-8°C
HRP-Conjugate reagent 3ml×1 bottle 6ml×1 bottle 2-8°C
Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8°C
Chromogen Solution A 3ml×1 bottle 6ml×1 bottle 2-8°C
Chromogen Solution B 3ml×1 bottle 6ml×1 bottle 2-8°C
Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8°C
Wash solution
(20ml×20 fold)
×1bottle
(20ml×30 fold)
×1bottle
2-8 ° C
Specimen requirements
1. serum- coagulation at room temperature 10-20 mins,centrifugation 20-min at the speed of
2000-3000 rpm remove supernatant, If precipitation appeared, Centrifugal again.
2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20
Mins ,centrifugation 20-min at the speed of 2000-3000 rpm remove supernatant, If
Precipitation appeared, Centrifugal again.
3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 rpm
Remove supernatant, If precipitation appeared, Centrifugal again. The Operation of
Hydrothorax and cerebrospinal fluid Reference to it.
4. cell culture supernatant-detect secretory components, collect sue a sterile container,
Centrifugation 20-min at the speed of 2000-3000 rpm remove supernatant,detect the
Composition of cells, Dilut cell suspension with PBS (pH 7.2-7.4), Cell concentration
Reached 1 million / ml, repeated freeze-thaw cycles, damage cells and release of
Intracellular components, centrifugation 20-min at the speed of 2000-3000 rpm remove
Superphosphate, If precipitation appeared, Centrifugal again.
5. Tissue samples- After cutting samples, check the weight,add PBS(PH7.2-7.4), Rapidly
Frozen with liquid nitrogen, maintain samples at 2-8°C after melting,add PBS(pH7.4),
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Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 rpm
Remove supernatant.
6. 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 °C to preserve, avoided repeated freeze-thaw cycles.
7. Can't detect the sample which contains 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 young well, then add standard dilution 50μl to the fifth and the sixth well, mix; take out 50μl
From the fifth and the sixth well 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(add Sample 50μl to
Each well after Diluting ,(density: 6ng/L, 4ng/L, 2ng/L, 1ng/L, 0.5ng/L)
2.add sample:Set blank wells separately (blank comparison wells don't add sample and
HRP-Conjugate reagent, other each step operation is same). testing sample well.
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.
3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37°C.
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.
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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°C
10.Stop the reaction:Add Stop Solution50μl to each well, Stop the reaction(the blue color
Change to yellow color).
11.assay:take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and
Within 15min.
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 mins, 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 ),plediment 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.
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.8