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Human Lipocalin-2/NGAL DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Volume Required
100 µL
Assay Range
78.1 - 5,000 pg/mL
Sufficient Materials
For five and fifteen 96-well plates*
Specificity
Please see the product datasheet

* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Lipocalin-2/NGAL. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

 

Product Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Economical alternative to complete kits

Kit Content

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.

The components listed above may be purchased separately:

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4

Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered

Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H2SO4 (Catalog # DY994)

Microplates: R&D Systems (Catalog # DY990)

Plate Sealers: ELISA Plate Sealers (Catalog # DY992)

Data Example

Human Lipocalin-2 / NGAL ELISA Standard Curve

Product Datasheets

Preparation and Storage

Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: Lipocalin-2/NGAL

The Lipocalin family comprises a diverse group of mostly secreted soluble proteins that bind hydrophobic ligands and act as transporters, carrying small molecules to specific cells. Lipocalins are related by possessing an 8-stranded beta-barrel structure. Lipocalin-1, also named tear lipocalin (TL), von Ebners gland protein (VEG) and tear pre-albumin, binds a large number of hydrophobic molecules and exhibits cysteine proteinase inhibitor and endonuclear activities. Lipocalin-2, also known as neutrophil gelatinase-associated lipocalin (NGAL), is a component of granules in neutrophils from tissues that are normally exposed to microorganisms and is upregulated during inflammation. Lipocalin-2 can form homodimers and can heterodimerize with the neutrophil gelatinase MMP-9.

Long Name:
Neutrophil Gelatinase-associated Lipocalin
Entrez Gene IDs:
3934 (Human); 16819 (Mouse); 170496 (Rat); 102137149 (Cynomolgus Monkey)
Alternate Names:
24p3; 25 kDa alpha-2-microglobulin-related subunit of MMP-9; HNL; LCN2; lipocalin 2 (oncogene 24p3); lipocalin 2; Lipocalin2; Lipocalin-2; migration-stimulating factor inhibitor; MSFI; neutrophil gelatinase-associated lipocalin; NGAL; NGALlipocalin-2; Oncogene 24p3; p25; Siderocalin; Uterocalin

Assay Procedure

GENERAL ELISA PROTOCOL

Plate Preparation

  1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
  2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

Citations for Human Lipocalin-2/NGAL DuoSet ELISA

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

18 Citations: Showing 1 - 10
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  1. Urine Hydrogen Peroxide Levels and Their Relation to Outcome in Patients with Sepsis, Septic Shock, and Major Burn Injury
    Authors: M Lipcsey, M Bergquist, R Sirén, A Larsson, F Huss, J Pravda, M Furebring, J Sjölin, H Janols
    Biomedicines, 2022;10(4):.
    Species: Human
    Sample Types: Plasma
  2. Serum and Urinary Neutrophil Gelatinase-Associated Lipocalin Are Not Associated With Serum Redox Parameters in Amateur Athletes After an Ultramarathon
    Authors: ACC Loureiro, GF Nocrato, ALL Correia, RS de Matos, JCCN Filho, EF Daher, FHM Pinto, AC de Oliveir, VM Ceccatto, RS Fortunato, DP de Carvalh
    Frontiers in Physiology, 2022;13(0):811514.
    Species: Human
    Sample Types: Serum
  3. Urinary cytokines correlate with acute kidney injury in critically ill COVID-19 patients
    Authors: A Gradin, H Andersson, T Luther, SB Anderberg, S Rubertsson, M Lipcsey, M Åberg, A Larsson, R Frithiof, M Hultström
    Cytokine, 2021;146(0):155589.
    Species: Human
    Sample Types: Urine
  4. Lipocalin 2 mediates appetite suppression during pancreatic cancer cachexia
    Authors: B Olson, X Zhu, MA Norgard, PR Levasseur, JT Butler, A Buenafe, KG Burfeind, KA Michaelis, KR Pelz, H Mendez, J Edwards, SM Krasnow, AJ Grossberg, DL Marks
    Nature Communications, 2021;12(1):2057.
    Species: Human
    Sample Types: Plasma
  5. Potential novel biomarkers for chronic lung allograft dysfunction and azithromycin responsive allograft dysfunction
    Authors: C Veraar, J Kliman, A Benazzo, F Oberndorfe, M Laggner, P Hacker, T Raunegger, S Janik, P Jaksch, W Klepetko, HJ Ankersmit, B Moser
    Scientific Reports, 2021;11(1):6799.
    Species: Human
    Sample Types: Serum
  6. In Vitro Characterization of Gut Microbiota-Derived Commensal Strains: Selection of Parabacteroides distasonis Strains Alleviating TNBS-Induced Colitis in Mice
    Authors: B Cuffaro, ALW Assohoun, D Boutillier, L Súkeníková, J Desramaut, S Boudebbouz, S Salomé-Des, J Hrdý, AJ Waligora-D, E Maguin, C Grangette
    Cells, 2020;9(9):.
    Species: Human
    Sample Types: Cell Culture Supernates
  7. Stabilization of surface-bound antibodies for ELISA based on a reversable zeolitic imidazolate framework-8 coating
    Authors: L Kang, S Smith, C Wang
    Journal of colloid and interface science, 2020;588(0):101-109.
    Species: Human
    Sample Types: Recombinant Protein
  8. TNFR1, TNFR2, neutrophil gelatinase-associated lipocalin and heparin binding protein in identifying sepsis and predicting outcome in an intensive care cohort
    Authors: M Bergquist, L Samuelsson, A Larsson, J Tydén, J Johansson, M Lipcsey
    Scientific Reports, 2020;10(1):15350.
    Species: Human
    Sample Types: Plasma
  9. Exploring the potential effect of paricalcitol on markers of inflammation in de novo renal transplant recipients
    Authors: HK Pihlstrøm, T Ueland, AE Michelsen, P Aukrust, F Gatti, C Hammarströ, M Kasprzycka, J Wang, G Haraldsen, G Mjøen, DO Dahle, K Midtvedt, IA Eide, A Hartmann, H Holdaas
    PLoS ONE, 2020;15(12):e0243759.
    Species: Human
    Sample Types: Plasma
  10. Topical Estrogen Treatment Augments the Vaginal Response to Escherichia coli Flagellin
    Authors: A Stanton, C Mowbray, M Lanz, K Brown, P Hilton, A Tyson-Capp, RS Pickard, ASM Ali, J Hall
    Sci Rep, 2020;10(1):8473.
    Species: Human
    Sample Types: Vaginal Fluid
  11. High levels of cell-free DNA accurately predict late acute kidney injury in patients after cardiac surgery
    Authors: J Merkle, A Daka, AC Deppe, T Wahlers, A Paunel-Gör
    PLoS ONE, 2019;14(6):e0218548.
    Species: Human
    Sample Types: Plasma
  12. Metformin alleviates nickel-induced autophagy and apoptosis via inhibition of hexokinase-2, activating lipocalin-2, in human bronchial epithelial cells
    Authors: YT Kang, WC Hsu, CH Wu, IL Hsin, PR Wu, KT Yeh, JL Ko
    Oncotarget, 2017;8(62):105536-105552.
    Species: Human
    Sample Types: Cell Culture Supernates
  13. Silk-based blood stabilization for diagnostics
    Proc Natl Acad Sci USA, 2016;113(21):5892-7.
    Species: Human
    Sample Types: Saliva
  14. Microbiota-inducible Innate Immune, Siderophore Binding Protein Lipocalin 2 is Critical for Intestinal Homeostasis
    Cell Mol Gastroenterol Hepatol, 2016;2(4):482-498.e6.
    Species: Human
    Sample Types: Cell Lysates
  15. Rapid renal alpha-1 antitrypsin gene induction in experimental and clinical acute kidney injury.
    Authors: Zager R, Johnson A, Frostad K
    PLoS ONE, 2014;9(5):e98380.
    Species: Human
    Sample Types: Urine
  16. Neisseria gonorrhoeae modulates iron-limiting innate immune defenses in macrophages.
    Authors: Zughaier, Susu M, Kandler, Justin L, Shafer, William
    PLoS ONE, 2014;9(1):e87688.
    Species: Human
    Sample Types: Cell Culture Supernates
  17. Effects of high-dose cholecalciferol on serum markers of inflammation and immunity in patients with early chronic kidney disease.
    Authors: Alvarez J, Zughaier S, Law J, Hao L, Wasse H, Ziegler T, Tangpricha V
    Eur J Clin Nutr, 2013;67(3):264-9.
    Species: Human
    Sample Types: Serum
  18. Lipocalin-2 increases fat oxidation in vitro and is correlated with energy expenditure in normal weight but not obese women.
    Authors: Paton C, Rogowski M, Kozimor A, Stevenson J, Chang H, Cooper J
    Obesity (Silver Spring), 2013;21(12):E640-8.
    Species: Human
    Sample Types: Plasma

FAQs

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Reviews for Human Lipocalin-2/NGAL DuoSet ELISA

Average Rating: 4.6 (Based on 5 Reviews)

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Human Lipocalin-2/NGAL DuoSet ELISA
By Anonymous on 04/02/2020
Sample Tested: Serum and Plasma

We used this kit for the quantification of Lipocalin 2 in human serum and plasma. Works very well and well-described protocol.


Human Lipocalin-2/NGAL DuoSet ELISA
By Alex Gonzalez on 12/05/2019
Sample Tested: Serum,Urine

Cost-effective and easy to use kit. Ran serum at 1:300 dilution and urine at 1:200 dilution.


Human Lipocalin-2/NGAL DuoSet ELISA
By Jessie Tweedie on 03/23/2018
Sample Tested: human fecal samples

Human Lipocalin-2/NGAL DuoSet ELISA
By Jonatan Dereke on 01/09/2018
Sample Tested: EDTA Plasma

We ran this analysis at a 1:200 dilution for human plasma samples with an inter-coefficient of variation of 3.5 - 6%.


Human Lipocalin-2/NGAL DuoSet ELISA
By Balaji Mahender on 12/20/2017
Sample Tested: EDTA Plasma