Manual of Resistin Elisa Kit

Resistin Elisa Kit Instructions This kit is for research use only.
Detection range: 96T
0μg / L -40μg / L
purpose of usage:
This kit is used to determine the content of Resistin in human serum, plasma and related liquid samples.
Experimental principle of human resistin (Resistin) Elisa kit instructions This kit uses the double antibody sandwich method to determine the level of human resistin (Resistin) in the specimen. Purified human resistin
(Resistin) antibody coated microwell plate to make solid phase antibody, then add resistin (Resistin) to the microwell coated with monoclonal antibody,
It is then combined with HRP-labeled Resistin antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. 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 Resistin in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human resistin (Resistin) in the sample was calculated by a standard curve.
Kit composition
1 30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle
2 Enzyme label reagent 6ml × 1 bottle 8 standard (80μg / L) 0.5ml × 1 bottle
3 Enzyme label coated plate 12 wells × 8 strips 9 standard dilutions 1.5ml × 1 bottle
4 Sample diluent 6ml × 1 bottle 10 instructions 1 copy
5 Developer A solution 6ml × 1 bottle 11 2 sealing film
6 Developer B solution 6ml × 1 / bottle 12 sealed bag 1 specimen requirement
1. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted 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
2. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
Steps
1. Dilution of standard products: This kit provides one original standard product. The user can perform dilution in a small test tube according to the following chart.
40μg / L No. 5 standard, 150μl original standard, add 150μl standard dilution
20μg / L No. 4 standard 150μl No. 5 standard added 150μl standard dilution
10μg / L No. 3 standard 150μl No. 4 standard added 150μl standard dilution
5μg / L No. 2 standard 150μl No. 3 standard added 150μl standard dilution
2.5μg / L No. 1 standard 150μl No. 2 standard added 150μl standard diluent
2. Add sample: set up blank wells separately (the blank control wells do not add samples and enzyme label reagents, the rest of the steps are the same), standard wells,
Sample hole to be tested. Add 50μl of the standard on the enzyme-coated plate accurately, and add 40μl of sample diluent to the sample well.
Then add 10μl of the sample to be tested (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 ° C for 30 minutes.
4. Mixing solution: dilute 20 times concentrated washing solution with distilled water 20 times and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds, then discard, repeat 5 times and pat dry.
6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.
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, and then add 50μl of developer B, mix gently, and develop color at 37 ℃ in the dark
15 minutes.
10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Summary of operating procedures:
The calculation takes the concentration of the standard as the abscissa and the OD value as the ordinate, and draws a standard curve on the coordinate paper.
The corresponding concentration of OD value is found from the standard curve; then 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 With a dilution factor,
This is the actual concentration of the sample.
Precautions
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 substance to be tested in the specimen is too high (the OD value of the sample 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. Please multiply the total dilution for the calculation Multiple (× 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.
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
Human Resistin
FOR RESEARCH USE ONLY
Assay range: 0μg / L -40μg / L 96 determinations
Purpose
This kit allows for the determination of Resistin concentrations in Human serum, cell
culture supernates and other biological fluids
Principle of the assay
The kit assay Human Resistin level in the sample, use Purified Human Resistin
antibody to coat microtiter plate wells, make solid-phase antibody, then add Resistin to wells,
Combined Resistin antibody which With HRP labeled goat anti-Human 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 Human Resistin in the
samples is then determined by comparing the OD 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 (80μg / 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
RD
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:
40μg / L 5 Standard 150μl Original density Standard + 150μl Standard diluent
20μg / L 4 Standard 150μl 5 Standard + 150μl Standard diluent
10μg / L 3 Standard 150μl 4 Standard + 150μl Standard diluent
5μg / L 2 Standard 150μl 3 Standard + 150μl Standard diluent
2.5μg / L 1 Standard 150μl 2 Standard + 150μl Standard diluent
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 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 ℃.
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 ℃
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.
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 human Resistin (Resistin) Elisa kit instructions

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