Mouse Very low density lipoprotein (VLDL)ELISA Kit instruction
Intended use
This VLDL ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of VLDL in the sample, this VLDL ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus VLDL αconcentration. The concentration of VLDL in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Sample collection and storages
1. Serum: Use non-pyrogenic and endotoxin in vitro, during the operation to avoid any cell stimulation, collection of blood, centrifuge 3000 rpm for 10 minutes will be carefully serum and red blood cells quickly separated.
2. Plasma: EDTA, citrate or heparin. 3000 rpm the supernatant centrifuged for 30 minutes.
3. supernatant: 3000 rpm centrifugation for 10 minutes to remove particles and polymers.
4. homogenate: The amount of saline organizations to join the broken. 3000 rpm the supernatant centrifuged for 10 minutes.
5. Save: If the sample is collected and is not detected in time, please press the amount of separate packaging, frozen at -20 ℃, avoid repeated freezing and thawing, thaw at room temperature and to ensure adequate sample evenly thawed.
Materials required but not supplied
1. Standard microplate reader(450nm)
2. Precision pipettes and Disposable pipette tips.
3. 37 ℃ incubator
Precautions
1. kit stored in 2-8 ℃, 20 minutes before use at room temperature balance. Remove from the refrigerator will be concentrated washing liquid crystal, which is a normal phenomenon in the water bath crystals compley dissolved before use.
2. experiments without immediay back into the ziplock bag of panel, the sealing (low temperature dried) to preserve.
3. Standard dilution can be regarded as a negative control or blank; pretreated samples without dilution, 10μL of sample can be obtained directly.
4. in strict accordance with the instructions indicated in the time, plus the order of fluid volume and incubation operations.
5. all liquid components Shake well before use.
Materials supplied
REAGENTS(store at2-8℃) | 1×96WELLS | 0.5×96WELLS | RECONSTTTUTION |
96/48-wells miorou | 12*8strips | 12*4strips | Ready-to-use |
Standard（300ng/ml） | 0.5ml | 0.5ml | Diluted according to instructions |
Standard diluent | 6.0ml | 3.0ml | Ready-to-use |
Sample diluent | 6.0ml | 3.0ml | Ready-to-use |
HRP-Conjugate reagent | 6.0ml | 3.0ml | Ready-to-use |
20X Wash solution | 20ml | 20ml | Diluted according to instructions |
Chromogen Solution A | 6.0ml | 3.0ml | Ready-to-use |
Chromogen Solution B | 6.0ml | 3.0ml | Ready-to-use |
Stop Solution | 6.0ml | 3.0ml | Ready-to-use |
Closure plate membrane | 2 | 2 | Ready-to-use |
User manual | 1 | 1 | Ready-to-use |
Sealed bags | 1 | 1 | Ready-to-use |
Note: Standard diluent with Standard diluent concentration was followed by: 300、150、75、37.5、18.7、0ng/ml
Reagent preparation
20 × dilution of washing buffer: distilled water, diluted by 1:20, or 1 copies of the 20 × washing buffer plus 19 copies of the distilled water
Washer Method
1.hand-washer: do dump hole liquid, washing liquid to fill each well, after the rejection of possible standing 1min hole liquid, pat dry on absorbent paper, such a washer 5.
2. Automatic Washer: Every hole into the lotion 350μL, soaking 1min, washe 5.
Steps
1. from room temperature after 20min foil bag balance required to strip out the remaining slab sealed with ziplock back 4 ℃.
2. Set standard bore holes and sample, standard hole plus different concentrations of each standard 50μL;
3. test sample test sample holes before adding 10μL, plus sample diluent 40μL;
4. then the sample standard bore holes and each hole by adding horseradish peroxidase (HRP) labeled detection antibody 50μL, sealed with the seal plate membrane pore reaction, 37 ℃ water bath or incubator and incubated for 60min.
5. Discard liquid, pat dry with absorbent paper, washing liquid to fill each hole, standing 1min, rejection to the washing liquid, absorbent paper, pat dry, repeat 5 times plate washer (also available washer and washer.)
6. each hole by adding the substrate A, B each 50μL, 37 ℃ dark incubated 15min.
7. each hole by adding stop solution 50μL, 15min, the measured wavelength at 450nm, OD value of the hole.
Performance Kit
1. Accuracy: standard linear regression correlation coefficient R with the expected value of the concentration, greater than equal to 0.9900.
2. Sensitivity: The minimum detectable concentration is less than 1.0.ng/ml
3. Specificity: not soluble structural analogues with other cross-reaction.
4. Repeatability: plate, the plate coefficient of variation was less than 15%.
5. Storage :2-8 ℃, dark moisture preservation.
6. Duration: 6 months
7.Assay range：9.3ng/ml- 300ng/ml
Results to determine
The standard curve: The Excel worksheet to standard concentration for the abscissa, corresponding to the OD value for the vertical axis and draw a standard linear regression curve, calculated by curve equation sample concentration.
Disclaimer
1. kit used for research purposes only and not for clinical trials or Mouse experiments, otherwise all the consequences arising from the experimenter commitment, the Company shall not be responsible.
2. in strict accordance with manufacturer's instructions, the experimenter violation of operating instructions, the consequences borne by the experimenter
