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This ELISA kit can be applied to the in vitro quantitative determination of Rat HPF4-Ab concentrations in serum, plasma and other biological fluids.
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antigen specific to Rat HPF4-Ab. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Rat HPF4-Ab and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. After incubation, free components are washed away. Then the substrate solution is added to each well, only those wells that contain Rat HPF4-Ab, biotinylated detection antigen and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and appears yellow in color. The optical density (OD) can be measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Rat HPF4-Ab. The concentration of Rat HPF4-Ab in samples can be calculated by comparing the OD of the samples to the standard curve.
|Detection Range||0.78—50 ng/mL|
|Sample Type||Serum, plasma and other biological fluids|
This kit recognizes Rat HPF4-Ab in samples. No significant cross-reactivity or interference was observed.
For convenience in result calculation, absorbance can be used as ordinate and standard concentrations as abscissa. The standard curve provided in the manual is only for reference, and experimenters should draw the standard curve based on data of themselves.
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Rat HPF4-Ab were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Rat HPF4-Ab were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||6.61||5.92||3.90||5.51||5.74||3.97|
The recovery of Rat HPF4-Ab spiked to three different levels in samples throughout the range of the assay in various matrices was evaluated.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||87-98||93|
Samples were spiked with high concentrations of Rat HPF4-Ab and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
|Serum (n=5)||EDTA plasma (n=5)|
The unopened kit can be stored at 4℃ for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions since the kit is received.
|Micro ELISA Plate(Dismountable)||8 wells ×12 strips||-20℃, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ag (100×)||1 vial, 120 μL|
|Concentrated HRP Conjugate (100×)||1 vial, 120 μL||-20℃(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||4℃(shading light), 6 months|
|Biotinylated Detection Ag Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25×)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||4℃, 6 months|
|Stop Solution||1 vial, 10 mL||4℃(shading light)|
|Plate Sealer||5 pieces|
|Certificate of Analysis||1 copy|
1.Add 100 μL of standard or sample to each well. Incubate for 90 min at 37℃.
2.Remove the liquid.
3.Add 100 μL of Biotinylated Detection Ag. Incubate 1 hour at 37℃. Aspirate and wash 3 times.
4.Add 100 μL HRP Conjugate. Incubate 30 min at 37℃.Aspirate and wash 5 times.
5.Add 90 μL of Substrate Reagent. Incubate for 15 min at 37℃.
6.Add 50 μL of Stop Solution.
7.Read the OD at 450 nm immediately. Calculation of the results.