Human CD14 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 CD14. 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: CD14
CD14 is a glycoprotein that mediates the interaction of lipopolysaccharide (LPS, endotoxin) with cells, thereby signaling the presence of gram-negative bacteria (1-3). CD14 is either soluble (CD14) (4, 5) or membrane-bound (mCD14) by a glycosylphosphatidylinositol (GPI) anchor (6, 7). mCD14 is a 55 kDa glycoprotein (1), while CD14 varies from about 43 to 53 kDa, depending on the degree of glycosylation and whether it was synthesized without the anchor or was shed by phospholipase cleavage of the anchor or by proteolysis (12-14). There is no evidence for different mRNAs for m- and CD14. There is no apparent sequence homology with other proteins. The sequence of human CD14 is 63-73% identical to that of mouse, rat, or rabbit CD14 (15).
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 CD14 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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Baseline circulating biomarkers, their changes, and subsequent suicidal ideation and depression severity at 6 months: A prospective analysis in patients with mood disorders
Authors: Lengvenyte, A;Cognasse, F;Hamzeh-Cognasse, H;Sénèque, M;Strumila, R;Olié, E;Courtet, P;
Psychoneuroendocrinology
Species: Human
Sample Types: Plasma
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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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Immunoglobulin G N-glycan markers of accelerated biological aging during chronic HIV infection
Authors: Giron, LB;Liu, Q;Adeniji, OS;Yin, X;Kannan, T;Ding, J;Lu, DY;Langan, S;Zhang, J;Azevedo, JLLC;Li, SH;Shalygin, S;Azadi, P;Hanna, DB;Ofotokun, I;Lazar, J;Fischl, MA;Haberlen, S;Macatangay, B;Adimora, AA;Jamieson, BD;Rinaldo, C;Merenstein, D;Roan, NR;Kutsch, O;Gange, S;Wolinsky, SM;Witt, MD;Post, WS;Kossenkov, A;Landay, AL;Frank, I;Tien, PC;Gross, R;Brown, TT;Abdel-Mohsen, M;
Nature communications
Species: Human
Sample Types: Plasma
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Predictors of HIV rebound differ by timing of antiretroviral therapy initiation
Authors: Li, JZ;Melberg, M;Kittilson, A;Abdel-Mohsen, M;Li, Y;Aga, E;Bosch, RJ;Wonderlich, ER;Kinslow, J;Giron, LB;Di Germanio, C;Pilkinton, M;MacLaren, L;Keefer, M;Fox, L;Barr, L;Acosta, E;Ananworanich, J;Coombs, R;Mellors, J;Deeks, S;Gandhi, RT;Busch, M;Landay, A;Macatangay, B;Smith, DM;AIDS Clinical Trials Group A5345 Study Team, ;
JCI insight
Species: Human
Sample Types: Plasma
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Toll-like Receptor 2 Mediated Immune Regulation in Simian Immunodeficiency Virus-Infected Rhesus Macaques
Authors: Boby, N;Williams, KM;Das, A;Pahar, B;
Vaccines
Species: Primate - Macaca mulatta (Rhesus Macaque)
Sample Types: Plasma
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Impaired gut microbiota-mediated short-chain fatty acid production precedes morbidity and mortality in people with HIV
Authors: Sereti, I;Verburgh, ML;Gifford, J;Lo, A;Boyd, A;Verheij, E;Verhoeven, A;Wit, FWNM;Schim van der Loeff, MF;Giera, M;Kootstra, NA;Reiss, P;Vujkovic-Cvijin, I;
Cell reports
Species: Human
Sample Types: Plasma
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Plasma Glycomic Markers of Accelerated Biological Aging During Chronic HIV Infection
Authors: Giron, LB;Liu, Q;Adeniji, OS;Yin, X;Kannan, T;Ding, J;Lu, DY;Langan, S;Zhang, J;Azevedo, JLLC;Hanna, DB;Ofotokun, I;Lazar, J;Fischl, MA;Haberlen, S;Macatangay, B;Adimora, AA;Jamieson, BD;Rinaldo, C;Merenstein, D;Roan, NR;Kutsch, O;Gange, S;Wolinsky, S;Witt, M;Post, WS;Kossenkov, A;Landay, A;Frank, I;Tien, PC;Gross, R;Brown, TT;Abdel-Mohsen, M;
bioRxiv : the preprint server for biology
Species: Human
Sample Types: Plasma
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Serum TGF-?1 and CD14 Predicts Response to Anti-TNF-? Therapy in IBD
Authors: Coufal, S;Kverka, M;Kreisinger, J;Thon, T;Rob, F;Kolar, M;Reiss, Z;Schierova, D;Kostovcikova, K;Roubalova, R;Bajer, L;Jackova, Z;Mihula, M;Drastich, P;Tresnak Hercogova, J;Novakova, M;Vasatko, M;Lukas, M;Tlaskalova-Hogenova, H;Jiraskova Zakostelska, Z;
Journal of immunology research
Species: Human
Sample Types: Serum
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Intestinal Barrier Dysfunction and Microbial Translocation in Patients with First-Diagnosed Atrial Fibrillation
Authors: L Blöbaum, M Witkowski, M Wegner, S Lammel, PA Schencke, K Jakobs, M Puccini, D Rei beta ner, D Steffens, U Landmesser, U Rauch, J Friebel
Biomedicines, 2023-01-10;11(1):.
Species: Human
Sample Types: Plasma
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Intestinal Injury Biomarkers Predict Mortality in Pediatric Severe Malaria
Authors: ML Sarangam, R Namazzi, D Datta, C Bond, CPB Vanderpool, RO Opoka, CC John, AL Conroy
MBio, 2022-09-07;0(0):e0132522.
Species: Human
Sample Types: Serum
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Immune responses in Omicron SARS-CoV-2 breakthrough infection in vaccinated adults
Authors: H Kared, AS Wolf, A Alirezayla, A Ravussin, G Solum, TT Tran, F Lund-Johan, JT Vaage, LS Nissen-Mey, UC Nygaard, O Hungnes, AH Robertson, LM Næss, L Trogstad, P Magnus, LA Munthe, S Mjaaland
Nature Communications, 2022-07-18;13(1):4165.
Species: Human
Sample Types: Plasma
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Irreversible depletion of intestinal CD4+ T-cells is associated with T-cell activation during chronic HIV infection
Authors: OE Asowata, A Singh, A Ngoepe, N Herbert, R Fardoos, K Reddy, Y Zungu, F Nene, N Mthabela, D Ramjit, F Karim, K Govender, T Ndung'u, JZ Porterfiel, JH Adamson, FG Madela, VT Manzini, F Anderson, A Leslie, HN Kløverpris
JCI Insight, 2021-11-22;0(0):.
Species: Human
Sample Types: Plasma
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Complement activation is associated with poor outcome after out-of-hospital cardiac arrest
Authors: V Chaban, ER Nakstad, H Stær-Jense, C Schjalm, I Seljeflot, J Vaage, C Lundqvist, JŠ Benth, K Sunde, TE Mollnes, GØ Andersen, SE Pischke
Resuscitation, 2021-06-11;0(0):.
Species: Human
Sample Types: Plasma
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Changes in liver steatosis in HIV-positive women are associated with the BMI, but not with biomarkers
Authors: R Fernandez-, MW Plankey, D Ware, J Bordon
Cytokine, 2021-05-12;0(0):155573.
Species: Human
Sample Types: Plasma
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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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Persistence of monocyte activation under treatment in people followed since acute HIV-1 infection relative to participants at high or low risk of HIV infection
Authors: S Novelli, C Lécuroux, C Goujard, J Reynes, A Villemant, L Blum, A Essat, V Avettand-F, O Launay, JM Molina, C Bourgeois, L Meyer
EBioMedicine, 2020-11-26;62(0):103129.
Species: Human
Sample Types: Plasma
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Expression of Matrix Metalloproteinases and Their Tissue Inhibitors in Peripheral Blood Leukocytes and Plasma of Children with Nonalcoholic Fatty Liver Disease
Authors: JB Trojanek, J Micha?kiew, R Grzywa-Czu, W Ja?czyk, L Gackowska, I Kubiszewsk, A Helmin-Bas, A Wierzbicka, M Szalecki, P Socha
Mediators Inflamm., 2020-09-10;2020(0):8327945.
Species: Human
Sample Types: Plasma
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Urinary proteomics links keratan sulfate degradation and lysosomal enzymes to early type 1 diabetes
Authors: JAD Van, S Clotet-Fre, AC Hauschild, I Batruch, I Jurisica, Y Elia, FH Mahmud, E Sochett, EP Diamandis, JW Scholey, A Konvalinka
PLoS ONE, 2020-05-26;15(5):e0233639.
Species: Human
Sample Types: Urine
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HIV-associated gut dysbiosis is independent of sexual practice and correlates with noncommunicable diseases
Authors: I Vujkovic-C, O Sortino, E Verheij, J Sklar, FW Wit, NA Kootstra, B Sellers, JM Brenchley, J Ananworani, MSV Loeff, Y Belkaid, P Reiss, I Sereti
Nat Commun, 2020-05-15;11(1):2448.
Species: Human
Sample Types: Plasma
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Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection
Authors: DM Muema, NA Akilimali, OC Ndumnego, SS Rasehlo, R Durgiah, DBA Ojwach, N Ismail, M Dong, A Moodley, KL Dong, ZM Ndhlovu, JM Mabuka, BD Walker, JK Mann, T Ndung'u
BMC Med, 2020-03-25;18(1):81.
Species: Human
Sample Types: Plasma
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Inflammatory Bowel Disease Types Differ in Markers of Inflammation, Gut Barrier and in Specific Anti-Bacterial Response
Authors: S Coufal, N Galanova, L Bajer, Z Gajdarova, D Schierova, Z Jiraskova, K Kostovciko, Z Jackova, Z Stehlikova, P Drastich, H Tlaskalova, M Kverka
Cells, 2019-07-13;8(7):.
Species: Human
Sample Types: Serum
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The effects of CD14 and IL-27 on induction of endotoxin tolerance in human monocytes and macrophages
Authors: C Petes, V Mintsopoul, RL Finnen, BW Banfield, K Gee
J. Biol. Chem., 2018-09-21;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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Soluble CD14 as a Diagnostic Biomarker for Smear-Negative HIV-Associated Tuberculosis
Authors: Y Liu, OC Ndumnego, T Chen, RS Kim, ER Jenny-Avit, T Ndung'u, D Wilson, JM Achkar
Pathogens, 2018-02-27;7(1):.
Species: Human
Sample Types: Serum
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Impact of early cART on HIV blood and semen compartments at the time of primary infection
Authors: A Chéret, C Durier, A Mélard, M Ploquin, J Heitzmann, C Lécuroux, V Avettand-F, L David, G Pialoux, JM Chennebaul, M Müller-Tru, C Goujard, C Rouzioux, L Meyer
PLoS ONE, 2017-07-14;12(7):e0180191.
Species: Human
Sample Types: Plasma
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Monocyte/macrophage and T cell activation markers are not independently associated with MI risk in healthy individuals - results from the HUNT Study
Authors: T Ueland, LE Laugsand, LJ Vatten, I Janszky, C Platou, AE Michelsen, JK Damås, P Aukrust, BO Åsvold
Int. J. Cardiol., 2017-06-12;0(0):.
Species: Human
Sample Types: Serum
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High Cellular Monocyte Activation in People Living With Human Immunodeficiency Virus on Combination Antiretroviral Therapy and Lifestyle-Matched Controls Is Associated With Greater Inflammation in Cerebrospinal Fluid
Authors: T Booiman, FW Wit, I Maurer, D De Frances, CA Sabin, AM Harskamp, M Prins, P Garagnani, C Pirazzini, C Franceschi, D Fuchs, M Gisslén, A Winston, P Reiss, NA Kootstra
Open Forum Infect Dis, 2017-05-25;4(3):ofx108.
Species: Human
Sample Types: Plasma
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Identification of promising plasma immune biomarkers to differentiate active pulmonary tuberculosis
Cytokine, 2016-08-31;88(0):99-107.
Species: Human
Sample Types: Plasma
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Intestinal cell damage and systemic immune activation in individuals reporting sensitivity to wheat in the absence of coeliac disease.
Authors: Uhde M, Ajamian M, Caio G, De Giorgio R, Indart A, Green P, Verna E, Volta U, Alaedini A
Gut, 2016-07-25;65(12):1930-1937.
Species: Human
Sample Types: Serum
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Associations between Cognition, Gender and Monocyte Activation among HIV Infected Individuals in Nigeria.
Authors: Royal W, Cherner M, Burdo T, Umlauf A, Letendre S, Jumare J, Abimiku A, Alabi P, Alkali N, Bwala S, Okwuasaba K, Eyzaguirre L, Akolo C, Guo M, Williams K, Blattner W
PLoS ONE, 2016-02-01;11(2):e0147182.
Species: Human
Sample Types: Plasma
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Plasma sCD14 as a biomarker to predict pulmonary exacerbations in cystic fibrosis.
Authors: Quon B, Ngan D, Wilcox P, Man S, Sin D
PLoS ONE, 2014-02-20;9(2):e89341.
Species: Human
Sample Types: Plasma
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Daily exposure to dust alters innate immunity.
Authors: Sahlander K, Larsson K, Palmberg L
PLoS ONE, 2012-02-15;7(2):e31646.
Species: Human
Sample Types: Serum
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Integrated proteomic analysis of human cancer cells and plasma from tumor bearing mice for ovarian cancer biomarker discovery.
Authors: Pitteri SJ, JeBailey L, Faca VM, Thorpe JD, Silva MA, Ireton RC, Horton MB, Wang H, Pruitt LC, Zhang Q, Cheng KH, Urban N, Hanash SM, Dinulescu DM
PLoS ONE, 2009-11-19;4(11):e7916.
Species: Human
Sample Types: Plasma
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Soluble CD14 and toll-like receptor-2 are potential salivary biomarkers for oral lichen planus and burning mouth syndrome.
Authors: Srinivasan M, Kodumudi KN, Zunt SL
Clin. Immunol., 2007-10-03;126(1):31-7.
Species: Human
Sample Types: Saliva
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Standard works also diluted in PBS only.
We used this kit for the quantification of CD14 in human serum and plasma. Works very well and well-described protocol.