Rat interleukin 6 (IL-6) ELISA kit instruction manual This reagent is for research purposes only: this kit is used to determine the content of interleukin 6 (IL-6) in rat serum, plasma and related liquid samples. Experimental principle: Kit composition: Instructions 1 copy 1 copy sealing film 2 pieces (48) 2 pieces (96) Standards: 180ng / L 0.5ml × 1 bottle 0.5ml × 1 bottle at 2-8 ℃ Store standard dilutions 1.5ml × 1 bottle 1.5ml × 1 bottle at 2-8 ℃ Store enzyme reagent 3 ml × 1 bottle 6 ml × 1 bottle at 2-8 ℃ to store sample dilution 3 ml × 1 bottle to 6 ml × 1 bottle at 2-8 ℃ to store developer A solution 3 ml × 1 bottle to 6 ml × 1 bottle at 2-8 ℃ to store developer B Liquid 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃ Storage stop solution 3ml × 1 bottle 6ml × 1 bottle 2-8 ℃ Store concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle 2-8 ℃ storage 2 Steps: Precautions: Calculation: Storage conditions and validity period: Eye Makeup,Mascara With Fibres,Single Eyebrow,False Eyelashes Suzhou Yimeijia368 Biological Technology Co.,Ltd. , https://www.yimeijiabeauty.com
This kit uses the double antibody sandwich method to determine the level of rat interleukin 6 (IL-6) in the specimen. The microplate was coated with purified rat interleukin 6 antibody to make a solid phase antibody. Interleukin 6 was added to the monoclonal antibody-coated microwells in turn, and then combined with HRP-labeled goat anti-mouse antibody to form an antibody -Antigen-enzyme-labeled antibody complex, after thorough washing, add substrate TMB to develop color. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with IL-6 in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rat interleukin 6 (IL-6) in the sample was calculated by a standard curve.
Sample processing and requirements:
1. Serum: Blood coagulates naturally at room temperature for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage.
2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation.
4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components in the cells, dilute the cell suspension with PBS (PH7.2-7.4), the cell kit consists of 48-well configuration, 96-well configuration and storage
Sealed bag 1 x 1 enzyme-coated plate 1 × 48 1 × 96 2-8 ℃ Store
The concentration reaches about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ℃. Add a certain amount of PBS (PH7.4) and homogenize the specimen by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use.
6. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to 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 ℃, but repeated freezing and thawing should be avoided.
7. The samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).
1. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of standard products in the first and second wells, and then add in the first and second wells Standard diluent 50 μl, mix well; then take 100 μl from the first and second wells and add them to the third and fourth wells respectively, then add standard dilutions to the third and fourth wells respectively 50 μl of solution, mix well; then take 50 μl of each in the third and fourth wells and discard, then add 50 μl of each to the fifth and sixth wells, and then in the fifth and sixth wells Add 50ul of standard dilution solution to the medium and mix well; after mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively 50 μl of standard diluent. After mixing, take 50 μl from the seventh and eighth wells and add them to the ninth and tenth wells. Then add 50 μl of the standard diluent to the ninth and tenth wells. After mixing, take 50 μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50 μl, and the concentrations are 120 ng / L, 80 ng / L, 40 ng / L, 20 ng / L, and 10 ng / L respectively).
2. Add samples: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40 μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10 μl of the test sample (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix.
3. Incubation: seal the plate with the sealing film and incubate at 37 ℃ for 30 minutes.
4. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing solution with distilled water 30 times (20 times of 48T) and then reserve.
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let stand for 30 seconds and then discard, repeat 5 times, pat dry.
6. Add enzyme: add 50 μl of enzyme label reagent to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: Add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ℃ in the dark for 15 minutes.
10. Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color turns to yellow at this time).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner zero and 4 50 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
1 . The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before use. If the enzyme-coated plate is unsealed after opening, the strip should be stored in a sealed bag.
2 . Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.
3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
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 test substance in the specimen is too high (the OD value of the sample 3 is greater than the OD value of the first well of the standard well), please dilute it with a certain multiple (n times) of the sample diluent and then determine it. Dilution factor (× n × 5).
5. The sealing film is limited to one-time use to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions, and the test results must be determined by the microplate reader.
8 . All samples, washing liquids and various wastes should be treated as infectious agents.
9 . The components of different batches of this reagent shall not be mixed.
10. If there is any difference with the English manual, the English manual shall prevail.
Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the graph paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; multiply by the dilution factor; or use the concentration of the standard Calculate the linear regression equation of the standard curve with the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply it by the dilution factor to obtain the actual concentration of the sample.
(This picture is for reference only)
Kit performance:
1. The correlation coefficient R between the linear regression of the sample and the expected concentration is more than 0.990.
2. In-batch and approval should be less than 9% and 11% respectively
examination range:
8ng / L -150ng / L
1. Store the kit: 2-8 ℃.
2. Validity: 6 months
FOR FOR FOR FOR RESEARCH RESEARCH RESEARCH RESEARCH USE USE USE USE ONLY ONLY ONLY ONLY4
Drug Drug Drug Drug Names Names Names Names
Generic Name: Rat Rat Rat Rat interleukin interleukin interleukin interleukin 6 6 6 6 (IL- (IL- (IL- (IL- (IL- 6 6 6 6)))) ELISA ELISA ELISA ELISA ELISA Kit Kit Kit Kit...
Purpose Purpose Purpose Purpose
This kit allows for the determination of IL-6 concentrations in Rat serum, blood plasma,
and other biological fluids.
Principle Principle Principle Principle of of of the of the the assay assay assay assay
T he kit assay Rat IL-6 level in the sample, use Purified Rat IL-6 to coat microtiter plate
wells, make solid-phase antibody, then add IL-6 to wells, Combined antibody which With HRP
labeled goat anti- mouse 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.
Materials Materials Materials provided provided provided provided with with with the the the the kkkk itititit
Rat Rat Rat Rat interleukin interleukin interleukin interleukin 6 6 6 65
Specimen Specimen Specimen Specimen requirements requirements requirements requirements
1. serum serum serum 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 plasma plasma 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 Urine Urine 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 cell cell cell culture culture culture culture supernatant supernatant supernatant 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 (PH7.2-7.4), 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 supernatant, If precipitation appeared, Centrifugal again.
5. Tissue Tissue Tissue Tissue samples samples samples samples-After cutting samples, check the weight, add PBS (PH7.2-7.4), Rapidly frozen with liquid nitrogen, maintain samples at 2-8 ℃ after melting, add PBS (PH7. 4), Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000r.pm Materials provided with the k it 48 determinations 96 determinations Storage User manual 1 1
Closure plate membrane 2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8 ℃
Standar d: 180ng / L 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ℃
Standard diluent 1.5ml × 1 bottle 1.5ml × 1 bottle 2-8 ℃
HRP-Conjugate reagent 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃
Sample diluent 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃
Chromogen Solution A 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃
Chromogen Solution B 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃
Stop Solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃
wash solution
(20ml × 20 fold)
× 1 bottle
(20ml × 30 fold)
× 1 bottle
2-8 ℃ 6
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 ℃ to preserve, Avoid repeated freeze -thaw cycles.
7. Can 't detect the sample which contain NaN3, because NaN3 inhibits HRP active. Assay Assay Assay Assay procedure procedure procedure procedure
1. Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100 μ l to the first and the second well, then add Standard dilution 50 μ l to the first and the second well, mix; take out 100 μ l form the first and the second well then add it to the third and the forth well separately. then add Standard dilution 50 μ l to the third and the forth well, mix; then take out 50 μ l from the third and the forth well discard, add 50 μ l to the fifth and the sixth well, then add Standard dilution 50 μ l to the fifth and the sixth well, mix; take out 50 μ l from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50 μ l to the seventh and the eighth well, mix; take out 50 μ l from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50 μ l to the ninth and the tenth well, mix, take out 50 μ l from the ninth and the tenth well discard (add Sample 50 μ l to each well after Diluting, (density: 120 ng / L, 80ng / L, 40 ng / L, 20ng / L, 10 ng / 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. Add Sample dilution 40 μ l to testing sample well, then add testing sample 10 μ l (sample final dilut ion is 5-
fold), add sample to well s, 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: 30-fold (or 2 0-fold) wash solution diluted 30-fold (or 2 0-fold) with distilled water and reserve.
5. Washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffe r to every well, still for 30s then drain, repeat 5 times, dry by pat.
6. add enzyme: Add HRP-Conjugate reagent 50 μ l to each well, except blank well.
7. incubate: Operation with 3.
8. washing: Operation with 5.7
9. color ： Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37 ℃
10. Stop the reaction ： Add Stop Solution 50 μ l 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 Important Important Important notes notes notes 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 s, if the number of sample is much, recommend to use Volley.
4. if the testing material content is excessively high er (The sample OD is bigger than the first standard well), please dilute Sample (n -fold), Please diluent e 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.
Calculate Calculate 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 sa mple OD value in the equation,
calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.
This This This This chartis chartis chartis chartis for for for reference reference reference reference only only only only8
Assay Assay Assay Assay range range range range
8ng / L -150ng / L
Storage Storage Storage Storage and and and and validity validity validity validity
1 . Storage: 2-8 ℃.
2 . validity: six months.