Human Lipocalin-2/NGAL DuoSet ELISA Summary
* 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)
Scientific Data
Product Datasheets
Preparation and Storage
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.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- 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.
- 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.
- Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
- 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.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- 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.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- 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.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- 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.
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Citations: Showing 1 - 10
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Identification and characterization of plasma proteins associated with intra-amniotic inflammation and/or infection in women with preterm labor.
Authors: Cho, HY;Lee, JE;Park, KH;Choi, BY;Lee, MJ;Jeong, DE;Shin, S;
Scientific reports
Species: Human
Sample Types: Plasma
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Urinary beta-2 microglobulin increases whereas TIMP-2 and IGFBP7 decline after unilateral nephrectomy in healthy kidney donors
Authors: de Rooij, ENM;Hoogeveen, EK;Romijn, FPHTM;van der Kooy, SW;Veighey, KV;Dekker, FW;van Kooten, C;Cobbaert, CM;de Fijter, JW;
Scientific reports
Species: Human
Sample Types: Urine
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Liraglutide and not lifestyle intervention reduces soluble CD163 after comparable weight loss in obese participants with prediabetes or type 2 diabetes mellitus
Authors: Grannes, H;Ueland, T;Simeone, P;Liani, R;Guagnano, MT;Aukrust, P;Michelsen, AE;Birkeland, K;di Castelnuovo, A;Cipollone, F;Consoli, A;Halvorsen, B;Gregersen, I;Santilli, F;
Cardiovascular diabetology
Species: Human
Sample Types: Plasma
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The Human Phospholipase B-II Precursor (HPLBII-P) in Urine as a Novel Biomarker of Glomerular Activity in COVID-19 and Diabetes Mellitus
Authors: Xu, S;Hultström, M;Larsson, A;Lipcsey, M;Lindskog, C;Bülow, S;Frithiof, R;Venge, P;
Journal of clinical medicine
Species: Human
Sample Types: Urine
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Systemic neutrophil degranulation and emergency granulopoiesis in patients with Clostridioides difficile infection
Authors: Ramakrishnan, G;Young, MK;Nayak, U;Rigo, I;Marrs, AS;Gilchrist, CA;Behm, BW;Madden, GR;Petri, WA;
Anaerobe
Species: Human
Sample Types: Plasma
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Potential Utility of Urinary Follistatin as a Non-Invasive Indicator of Acute Tubular Damage in Patients with Acute Kidney Injury
Authors: Nagayama, I;Takayanagi, K;Nagata, D;Hasegawa, H;Maeshima, A;
Cells
Species: Human
Sample Types: Urine
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Serum Lipocalin-2 Levels as a Biomarker in Pre- and Post-Pubertal Klinefelter Syndrome Patients: A Pilot Study
Authors: Paparella, R;Ferraguti, G;Fiore, M;Menghi, M;Micangeli, G;Tarani, F;Ligotino, A;Messina, MP;Ceccanti, M;Minni, A;Barbato, C;Lucarelli, M;Tarani, L;Petrella, C;
International journal of molecular sciences
Species: Human
Sample Types:
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Longitudinal Follow-Up of Serum and Urine Biomarkers Indicative of COVID-19-Associated Acute Kidney Injury: Diagnostic and Prognostic Impacts
Authors: Lablad, Y;Vanhomwegen, C;De Prez, E;Antoine, MH;Hasan, S;Baudoux, T;Nortier, J;
International journal of molecular sciences
Species: Human
Sample Types: Plasma, Urine
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Intravenous versus oral hydration to reduce the risk of postcontrast acute kidney injury after intravenous contrast-enhanced CT in patients with severe chronic kidney disease (ENRICH): a study protocol for a single-centre, parallel-group, open-labelled non-inferiority randomised controlled trial in Denmark
Authors: Ravn, EJ;Hasific, S;Thomassen, M;Hjortebjerg, R;Bach Laursen, K;Diederichsen, A;Bistrup, C;Øvrehus, KA;
BMJ open
Species: Human
Sample Types: Plasma, Urine
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No impact of a high-fat meal coupled with intermittent hypoxemia on acute kidney injury biomarkers in adults with and without obstructive sleep apnea
Authors: Goulet, N;Tetzlaff, EJ;Morin, R;Mauger, JF;Amaratunga, R;Kenny, GP;Imbeault, P;
Physiological reports
Species: Human
Sample Types: Plasma
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Identification of Potential Biomarkers for Cancer Cachexia and Anti-Fn14 Therapy
Authors: Z Cao, IJ Burvenich, K Zhao, C Senko, J Glab, R Fogliaro, Z Liu, I Jose, H Puthalakat, NJ Hoogenraad, LD Osellame, AM Scott
Cancers, 2022-11-10;14(22):.
Species: Human
Sample Types: Cell Culture Supernates
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Antenatal maternal depression, early life inflammation and neurodevelopment in a South African birth cohort
Authors: PJW Naudé, C Pariante, N Hoffman, SM Koopowitz, KA Donald, HJ Zar, DJ Stein
Brain, Behavior, and Immunity, 2022-07-06;105(0):160-168.
Species: Human
Sample Types: Serum
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Impact of Zinc to Copper Ratio and Lipocalin 2 in Obese Patients Undergoing Sleeve Gastrectomy
Authors: HM Demerdash, AA Sabry, OE Arida
Oncogene, 2022-06-10;2022(0):9278531.
Species: Human
Sample Types: Serum
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No association between intravenous fluid volume and endothelial glycocalyx shedding in patients undergoing resuscitation for sepsis in the emergency department
Authors: S Macdonald, E Bosio, NI Shapiro, L Balmer, S Burrows, M Hibbs, T Jowitt, L Smart, G Arendts, D Fatovich
Scientific Reports, 2022-05-24;12(1):8733.
Species: Human
Sample Types: Serum
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Increased Plasma Lipocalin-2 Levels in Patients with Myelin Oligodendrocyte Glycoprotein-IgG-Positive Optic Neuritis
Authors: JH Kim, H Lee, J Oh, K Suk, BY Chun
Journal of Clinical Medicine, 2022-05-07;11(9):.
Species: Human
Sample Types: Plasma
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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, 2022-04-05;10(4):.
Species: Human
Sample Types: Plasma
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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, 2022-03-17;13(0):811514.
Species: Human
Sample Types: Serum
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Antibody microarray analysis of amniotic fluid proteomes in women with cervical insufficiency and short cervix, and their association with pregnancy latency length
Authors: S Hong, KH Park, YE Lee, JE Lee, YM Kim, E Joo, I Cho
PLoS ONE, 2022-02-07;17(2):e0263586.
Species: Human
Sample Types: Amniotic Fluid
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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, 2021-05-24;146(0):155589.
Species: Human
Sample Types: Urine
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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, 2021-04-06;12(1):2057.
Species: Human
Sample Types: Plasma
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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, 2021-03-24;11(1):6799.
Species: Human
Sample Types: Serum
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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, 2020-12-24;588(0):101-109.
Species: Human
Sample Types: Recombinant Protein
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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, 2020-12-16;15(12):e0243759.
Species: Human
Sample Types: Plasma
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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, 2020-09-18;10(1):15350.
Species: Human
Sample Types: Plasma
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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, 2020-09-16;9(9):.
Species: Human
Sample Types: Cell Culture Supernates
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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, 2020-05-21;10(1):8473.
Species: Human
Sample Types: Vaginal Fluid
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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, 2019-06-18;14(6):e0218548.
Species: Human
Sample Types: Plasma
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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, 2017-11-06;8(62):105536-105552.
Species: Human
Sample Types: Cell Culture Supernates
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Microbiota-inducible Innate Immune, Siderophore Binding Protein Lipocalin 2 is Critical for Intestinal Homeostasis
Cell Mol Gastroenterol Hepatol, 2016-07-01;2(4):482-498.e6.
Species: Human
Sample Types: Cell Lysates
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Silk-based blood stabilization for diagnostics
Proc Natl Acad Sci USA, 2016-05-09;113(21):5892-7.
Species: Human
Sample Types: Saliva
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Rapid renal alpha-1 antitrypsin gene induction in experimental and clinical acute kidney injury.
Authors: Zager R, Johnson A, Frostad K
PLoS ONE, 2014-05-21;9(5):e98380.
Species: Human
Sample Types: Urine
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Neisseria gonorrhoeae modulates iron-limiting innate immune defenses in macrophages.
Authors: Zughaier, Susu M, Kandler, Justin L, Shafer, William
PLoS ONE, 2014-01-28;9(1):e87688.
Species: Human
Sample Types: Cell Culture Supernates
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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), 2013-07-29;21(12):E640-8.
Species: Human
Sample Types: Plasma
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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, 2013-01-30;67(3):264-9.
Species: Human
Sample Types: Serum
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Average Rating: 4.6 (Based on 5 Reviews)
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We used this kit for the quantification of Lipocalin 2 in human serum and plasma. Works very well and well-described protocol.
Cost-effective and easy to use kit. Ran serum at 1:300 dilution and urine at 1:200 dilution.
We ran this analysis at a 1:200 dilution for human plasma samples with an inter-coefficient of variation of 3.5 - 6%.