Instruction Manual for Rat Very Low Density Lipoprotein (VLDL) ELISA Kit
This kit is for research use only.
Detection range: 96T
3μg / ml -120μg / ml
purpose of usage:
This kit is used to determine the content of very low density lipoprotein (VLDL) in rat serum, plasma and related liquid samples.
Experimental principle
This kit uses the double antibody sandwich method to determine the level of rat very low density lipoprotein (VLDL) in the specimen. Microporous plates were coated with purified rat very low density lipoprotein (VLDL) antibody to make solid phase antibodies. Very low density lipoprotein (VLDL) was added to the microwells coated with mAb in turn, followed by Very low-density lipoprotein (VLDL) antibodies combine to form antibody-antigen-enzyme-labeled antibody complexes. After thorough washing, 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 the very low density lipoprotein (VLDL) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rat very low density lipoprotein (VLDL) 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 reagent
6ml × 1 bottle
8
Standard product (240μg / ml)
0.5ml × 1 bottle
3
Enzyme coated plate
12 holes × 8
9
Standard dilution
1.5ml × 1 bottle
4
Sample diluent
6ml × 1 bottle
10
Instructions
1 serving
5
Developer A liquid
6ml × 1 bottle
11
Sealing film
2 sheets
6
Developer B liquid
6ml × 1 / bottle
12
sealed bag
1
Specimen requirements
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. The 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 small test tubes according to the following chart.
120μg / ml
Standard No. 5
150μl of original standard is added to 150μl standard dilution
60μg / ml
Standard 4
150μl Standard No. 5 is added to 150μl standard dilution
30μg / ml
Standard 3
150μl Standard No. 4 is added to 150μl Standard Diluent
15μg / ml
Standard No. 2
150μl Standard No. 3 is added to 150μl Standard Diluent
7.5μg / ml
Standard 1
150μl Standard No. 2 is added to 150μl Standard Diluent
2. Sample addition: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), standard wells, sample wells to be tested. Accurately add 50μl of the standard on the enzyme-coated plate, add 40μl of sample diluent to the sample well, and 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 a sealing plate and incubate at 37 ° C for 30 minutes.
4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 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 and then discard, repeat this 5 times, 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, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 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 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Summary of operating procedures:
Calculation
Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply it 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.
Precautions
1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, 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 is greater than the OD value of the first well of the standard well), please first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution 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
RD
FOR RESEARCH USE ONLY
Assay range: 3μg / mL – 120μg / mL 96determinations
Purpose
This kit allows for the determination of VLDL concentrations in Ratserum, cellculture supernates and other biological fluids
Principle of the assay
The kit assay RatVLDLlevel in the sample, use Purified RatVLDLantibody to coat microtiter plate wells, make solid-phase antibody, then addVLDLto wells, CombinedVLDL 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 RatVLDLin 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 (240μg / ml)
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
1. extractas 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:
120μg / ml
5 Standard
150μl Original density Standard + 150μl Standard diluent
80μg / mL
4 Standard
150μl 5 Standard + 150μl Standard diluent
40μg / mL
3 Standard
150μl 4 Standard + 150μl Standard diluent
20μg / mL
2 Standard
150μl 3 Standard + 150μl Standard diluent
10μg / mL
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, discardLiquid, 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
AddStandard, Sample diluent, incubate for 30 min at 37 ℃.
Wash 5 time, AddHRP-Conjugate reagent, incubate for 30 min at 37 ℃.
Wash 5 times, Add Chromogen Solution A and B, incubate for 30 min at 37 ℃.
AddStopp 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 diluenteand 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
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