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Human NGAL(Neutrophil Gelatinase Associated Lipocalin) ELISA Kit

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Price:   $609
Cat Number:  E-EL-H0096
Lead Time:   7~10 days

Reactivity:  Human

Detection Range:  0.16~10 ng/mL

Sensitivity:  0.10 ng/mL

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Intended use

This ELISA kit applies to the in vitro quantitative determination of Human NGAL concentrations in serum, plasma and other biological fluids.

Test principle

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. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human NGAL and 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 nm ± 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.

Assay type Sandwich-ELISA
Format 96T/48T
Assay time 3.5h
Reactivity Human
Detection Method Colormetric
Detection Range 0.16—10 ng/mL
Sensitivity 0.10 ng/mL
Sample Volume Required Per Well 100μL
Sample Type serum, plasma and other biological fluids

Specificity

This kit recognizes Human NGAL in samples. No significant cross-reactivity or interference between Human NGAL and analogues was observed.

Typical data

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.

Concentration
(ng/mL)
O.D Average Corrected
10 2.252
2.306
2.279 2.217
5 1.496
1.554
1.525 1.463
2.5 0.863
0.863
0.863 0.801
1.25 0.41
0.432
0.421 0.359
0.63 0.255
0.245
0.25 0.188
0.32 0.168
0.142
0.155 0.093
0.16 0.108
0.112
0.11 0.048
0 0.062
0.062
0.062 --

Precision

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
Sample 1 2 3 1 2 3
n 20 20 20 20 20 20
Mean (ng/mL) 0.54 1.22 4.73 0.53 1.29 4.99
Standard deviation 0.03 0.05 0.26 0.04 0.07 0.23
C V (%) 5.56 4.10 5.50 7.55 5.43 4.61

Recovery

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 (%)
Serum (n=5) 85-101 92
EDTA plasma (n=5) 95-111 101
Cell culture media (n=5) 87-102 94

Linearity

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=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2 Range (%) 93-105 88-98 98-113
Average (%) 100 93 103
1:4 Range (%) 88-101 84-95 81-96
Average (%) 95 89 88
1:8 Range (%) 91-101 79-91 89-104
Average (%) 96 85 95
1:16 Range (%) 90-101 85-97 90-102
Average (%) 96 90 95

Kit components & Storage

An 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 once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable) 8 wells ×12 strips -20℃, 6 months
Reference Standard 2 vials
Concentrated Biotinylated Detection Ab (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℃, 6 months
Biotinylated Detection Ab 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℃(shading light)
Stop Solution 1 vial, 10 mL 4℃
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

  • Microplate reader with 450 nm wavelength filter
  • High-precision transfer pipette, EP tubes and disposable pipette tips
  • Incubator capable of maintaining 37℃
  • Deionized or distilled water
  • Absorbent paper
  • Loading slot for Wash Buffer

Assay Procedure

1.Add 100 μL standard or sample to each well. Incubate for 90 min at 37℃.

2.Remove the liquid.

3.Add 100 μL Biotinylated Detection Ab. Incubate for 1 hour at 37℃. Aspirate and wash 3 times.

4.Add 100 μL HRP Conjugate. Incubate for 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 Stop Solution.

7.Read at 450 nm immediately. Calculation of results.

Citations

  1. Publication: Ceylan H ?, Sayg?n ?, ?zel Türkcü ü. Assessment of acute aerobic exercise in the morning versus evening on asprosin, spexin, lipocalin-2, and insulin level in overweight/obese versus normal weight adult men[J]. Chronobiology International, 2020: 1-17.
    Sample Type: Serum
  2. Publication: Li H, Xu Q, Wang Y, et al. Serum neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for predicting high dose methotrexate associated acute kidney injury in children with acute lymphoblastic leukemia[J]. Cancer Chemotherapy and Pharmacology, 2020, 85(1): 95-103.
    Sample Type: Serum
  3. Publication: Nour I, Elmaghraby R, Shehata R, et al. Selective head cooling and acute kidney injury in neonates with hypoxic ischemic encephalopathy[J]. Journal of Neonatal-Perinatal Medicine, 2020 (Preprint): 1-10.
    Sample Type: Serum
  4. Publication: Sueud T, Hadi N R, Abdulameer R, et al. Assessing urinary levels of IL-18, NGAL and albumin creatinine ratio in patients with diabetic nephropathy[J]. Diabetes & Metabolic Syndrome: Clinical Research & Reviews, 2019, 13(1): 564-568.
    Sample Type: Urine
  5. Publication: Rejeki M S, Arozal W, Setiabudy R, et al. Evaluation of Kidney Injury Molecule-1 (KIM-1) and Neutrophil Gelatinase-Associated Lipocalin (NGAL) Urinary Levels for Detecting Kidney Dysfunction in Patients With Nasopharyngeal Cancer Treated With Cisplatin-Based Treatment[J]. Indonesian Journal of Cancer, 2018, 12(2): 60-66.
    Sample Type: Urine
  6. Publication: Bongers C C W G, Alsady M, Nijenhuis T, et al. Impact of acute versus prolonged exercise and dehydration on kidney function and injury[J]. Physiological reports, 2018, 6(11): e13734.
    Sample Type: Urine
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