QuicKey Human NGAL(Neutrophil Gelatinase Associated Lipocalin) ELISA Kit
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|Assay time||2.5 h|
|Sample type||serum, plasma, urine|
|Specificity||This kit recognizes Human NGAL in samples. No significant cross-reactivity or interference between Human NGAL and analogues was observed.|
|Reproducibility||Both intra-CV and inter-CV are < 10%.|
|Application||This ELISA kit applies to the in vitro quantitative determination of Human NGAL concentrations in serum, plasma, urine. Please consult technical support for the applicability if other biological fluids need to be tested.|
This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human NGAL. Samples (or Standards) and biotinylated detection antibody specific for Human NGAL are added to the micro ELISA plate wells. Human NGAL would combine with the specific antibody. Then Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human NGAL, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 ± 2 nm. The OD value is proportional to the concentration of Human NGAL. You can calculate the concentration of Human NGAL in the samples by comparing the OD of the samples to the standard curve.
Kit components & Storage
An unopened kit can be stored at 2-8℃ for six months. After test, the unused wells and reagents should be stored according to the table below. (The specific components might be slightly different in delivered kits, please refer to the manual for more information.)
|Item||Specifications||Storage conditions after test|
|Micro ELISA Plate (Dismountable)||
96T: 8 wells ×12 strips
48T: 8 wells ×6 strips
|2-8℃, 1 month|
96T: 2 vials
48T: 1 vial
|Discard unused reconstituted standard and dilutions|
|Reference Standard & Sample Diluent||1 vial, 20 mL||2-8℃|
|Biotinylated Detection Ab Working Solution||1 vial, 6 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25×)||1 vial, 30 mL|
|Concentrated HRP Conjugate (100×)||
96T: 1 vial, 120 μL
48T: 1 vial, 60 μL
|2-8℃ (Protect from light)|
|Substrate Reagent||1 vial, 10 mL|
|Stop Solution||1 vial, 10 mL||2-8℃|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Note: All reagent bottle caps must be tightened to prevent evaporation and microbial pollution. The volume of reagents in partial shipments is a little more than the volume marked on the label, please use accurate measuring equipment instead of directly pouring into the vial(s).
Other supplies required
1. Microplate reader with 450nm wavelength filter
2. High-precision transfer pipette, EP tubes and disposable pipette tips
3. Incubator capable of maintaining 37℃
4. Deionized or distilled water
5. Absorbent paper
6. Loading slot for Wash Buffer
Typical Standard Curve
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NGAL were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NGAL were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
The recovery of Human NGAL spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
Samples were spiked with high concentrations of Human NGAL and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
|Serum(n=4)||EDTA plasma (n=4)||Urine(n=4)|
LCN2, Lipocalin 2, Oncogene 24p3, MSFI
|1. Add 50μL standard or sample to the wells, immediately add 50μL Biotinylated Detection Ab working solution to each well. Incubate for 90 min at 37°C|
|2. Aspirate and wash the plate for 3 times|
|3. Add 100μL HRP conjugate working solution. Incubate for 30 min at 37°C. Aspirate and wash the plate for 5 times|
|4. Add 90μL Substrate Reagent. Incubate for 15 min at 37°C|
|5. Add 50μL Stop Solution|
|6. Read the plate at 450nm immediately. Calculation of the results|