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This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human MIP-1β . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human MIP-1β and Avidin-Horseradish Peroxidase (HRP) conjugate is added to each micro plate well successively and then incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human MIP-1β , biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color turns to yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human MIP-1β . You can calculate the concentration of Human MIP-1β in the samples by comparing the OD of the samples to the standard curve.
|Assay type||Sandwich ELISA|
|Sample Volume Required Per Well||100μL|
|Sample Type||serum, plasma and other biological fluids|
|Micro ELISA Plate||8 wells ×12 strips||4℃/-20℃#|
|Reference Standard||2 vials||4℃/-20℃#|
|Reference Standard & Sample Diluent||1vial 20mL||4℃|
|Concentrated Biotinylated Detection Ab||1vial 120μL||4℃/-20℃#|
|Biotinylated Detection Ab Diluent||1vial 10mL||4℃|
|Concentrated HRP Conjugate||1vial 120μL||4℃(shading light)|
|HRP Conjugate Diluent||1vial 10mL||4℃|
|Concentrated Wash Buffer (25×)||1vial 30mL||4℃|
|Substrate Reagent||1vial 10mL||4℃(shading light)|
|Stop Solution||1vial 10mL||4℃|
|Certificate of Analysis||1 copy|
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Human MIP-1β were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Human MIP-1β were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||5.800||5.100||5.100||5.300||5.400||5.500|
The recovery of Human MIP-1β 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)||87-100||95.000|
|Cell culture media (n=5)||88-104||95.000|
Samples were spiked with high concentrations of Human MIP-1β 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)||Cell culture media (n=5)|
This kit recognizes recombinant and natural Human MIP-1β . No significant cross-reactivity or interference was observed.
- Add 100μL standard or sample to each well. Incubate 90 mintues at 37℃.
- Add 100μL Biotinylated Detection Ab. Incubate 1 hour at 37℃.
- Aspirate and wash 3 times.
- Add 100μL HRP Conjugate. Incubate 30 minutes at 37℃.
- Aspirate and wash 5 times.
- Add 90μL Substrate Reagent. Incubate 15 minutes at 37℃.
- Add 50μL Stop Solution. Read it at 450nm immediately.
- Calculation of results.
All the reagents in the kit should be stored according to the labels on vials. Unused wells should be returned to the foil pouch with the desiccant pack and resealed along entire edge of zip-seal. Substrate Reagent shouldn't be kept at -20℃ (Check!). Exposure of reagents to strong light should be avoided in the process of incubation and storage. All the taps of reagents should be tightened to prevent evaporation and microbial contamination.
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