Mouse Complement Component C5a DuoSet ELISA
Mouse Complement Component C5a 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 mouse Complement ComponentC5a. 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: Complement Component C5a
Mouse Complement 5a (C5a) is an enzymatically generated glycoprotein that belongs to a family of structurally and functionally related proteins known as Anaphylatoxins. C5a is a 77 amino acid (aa) peptide that is created by the activity of C5a convertase on the C5 alpha -chain. Mouse C5a has four alpha -helices, plus three intrachain disulfide bonds that create a triple loop structure. In serum, proteolytic processing removes the C-terminal arginine, creating a low activity C5a desArg77 molecule. Mouse C5a shares 60% and 82% aa sequence identity to human and rat C5a, respectively. C5a binds to a signaling G-protein coupled receptor (GPCR) (C5aR/CD88), inducing neutrophil chemotaxis and endothelial cell activation. It also triggers an oxidative burst in macrophages and neutrophils, and induces release of histamine in basophils and mast cells. Alternatively, it may also bind to a nonsignaling GPCR termed C5L2 whose function is yet to be determined.
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 Mouse Complement Component C5a 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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The role of plasminogen activator inhibitor-2 in pneumococcal meningitis
Authors: NC Teske, JY Engelen-Le, S Dyckhoff-S, HW Pfister, M Klein, D van de Bee, CK Kirschning, U Koedel, MC Brouwer
Acta neuropathologica communications, 2022-10-29;10(1):155.
Species: Mouse
Sample Types: Tissue Homogenates
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CD55 Facilitates Immune Evasion by Borrelia crocidurae, an Agent of Relapsing Fever
Authors: G Arora, GE Lynn, X Tang, CE Rosen, D Hoornstra, A Sajid, JW Hovius, NW Palm, AM Ring, E Fikrig
MBio, 2022-08-29;0(0):e0116122.
Species: Mouse
Sample Types: Serum
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Complement activation contributes to subretinal fibrosis through the induction of epithelial-to-mesenchymal transition (EMT) in retinal pigment epithelial cells
Authors: M Llorián-Sa, EM Byrne, M Szczepan, K Little, M Chen, H Xu
Journal of Neuroinflammation, 2022-07-14;19(1):182.
Species: Mouse
Sample Types: Cell Culture Supernates
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Modulation of C5a-C5aR1 signaling alters the dynamics of AD progression
Authors: K Carvalho, ND Schartz, G Balderrama, HY Liang, SH Chu, P Selvan, A Gomez-Arbo, TJ Petrisko, MI Fonseca, A Mortazavi, AJ Tenner
Journal of Neuroinflammation, 2022-07-11;19(1):178.
Species: Mouse
Sample Types: Plasma
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Intracellular complement C5a/C5aR1 stabilizes beta-catenin to promote colorectal tumorigenesis
Authors: P Ding, Y Xu, L Li, X Lv, L Li, J Chen, D Zhou, X Wang, Q Wang, W Zhang, T Liao, QH Ji, QY Lei, W Hu
Cell Reports, 2022-05-31;39(9):110851.
Species: Mouse
Sample Types: Tissue Homogenates
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Endothelial expression of human amyloid precursor protein leads to amyloid beta in the blood and induces cerebral amyloid angiopathy in knock-in mice
Authors: Y Tachida, S Miura, Y Muto, H Takuwa, N Sahara, A Shindo, Y Matsuba, T Saito, N Taniguchi, Y Kawaguchi, H Tomimoto, T Saido, S Kitazume
The Journal of Biological Chemistry, 2022-03-31;0(0):101880.
Species: Mouse
Sample Types: Serum
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A potent truncated form of human soluble CR1 is protective in a mouse model of renal ischemia-reperfusion injury
Authors: AK Bongoni, IB Vikstrom, JL McRae, EJ Salvaris, N Fisicaro, MJ Pearse, S Wymann, T Rowe, AB Morelli, MP Hardy, PJ Cowan
Scientific Reports, 2021-11-08;11(1):21873.
Species: Mouse
Sample Types: Serum
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The impact of airborne endotoxin exposure on rheumatoid arthritis-related joint damage, autoantigen expression, autoimmunity, and lung disease
Authors: TR Mikuls, R Gaurav, GM Thiele, BR England, MG Wolfe, BP Shaw, KL Bailey, TA Wyatt, AJ Nelson, MJ Duryee, CD Hunter, D Wang, DJ Romberger, DP Ascherman, JA Poole
International immunopharmacology, 2021-08-27;100(0):108069.
Species: Mouse
Sample Types: Tissue Homogenates
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Serum amyloid P component is an essential element of resistance against Aspergillus fumigatus
Authors: A Doni, R Parente, I Laface, E Magrini, C Cunha, FS Colombo, JF Lacerda, A Campos, SN Mapelli, F Petroni, R Porte, T Schorn, A Inforzato, T Mercier, K Lagrou, J Maertens, JD Lambris, B Bottazzi, C Garlanda, M Botto, A Carvalho, A Mantovani
Nature Communications, 2021-06-18;12(1):3739.
Species: Mouse
Sample Types: BALF
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Evaluation of the gut microbiome in association with biological signatures of inflammation in murine polytrauma and shock
Authors: SA Appiah, CL Foxx, D Langgartne, A Palmer, CA Zambrano, S Braumüller, EJ Schaefer, U Wachter, BL Elam, P Radermache, CE Stamper, JD Heinze, SN Salazar, AK Luthens, AL Arnold, SO Reber, M Huber-Lang, CA Lowry, R Halbgebaue
Scientific Reports, 2021-03-23;11(1):6665.
Species: Mouse
Sample Types: Tissue Homogenates
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Host syndecan-1 promotes listeriosis by inhibiting intravascular neutrophil extracellular traps
Authors: RS Aquino, A Hayashida, PW Park
PLoS Pathog., 2020-05-26;16(5):e1008497.
Species: Mouse
Sample Types: Tissue Homogenates
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Complement receptor C3aR1 controls neutrophil mobilization following spinal cord injury through physiological antagonism of CXCR2
Authors: FH Brennan, T Jogia, ER Gillespie, LV Blomster, XX Li, B Nowlan, GM Williams, E Jacobson, GW Osborne, FA Meunier, SM Taylor, KE Campbell, KP MacDonald, JP Levesque, TM Woodruff, MJ Ruitenberg
JCI Insight, 2019-05-02;4(9):.
Species: Mouse
Sample Types: Tissue Homogenates
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Plasma protein adsorption on Fe3O4-PEG nanoparticles activates the complement system and induces an inflammatory response
Authors: V Escamilla-, A Solorio-Ro, M Uribe-Ramí, O Lozano, S Lucas, A Chagolla-L, R Winkler, A De Vizcaya
Int J Nanomedicine, 2019-03-25;14(0):2055-2067.
Species: Mouse
Sample Types: Plasma
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Complement Inhibitor CRIg/FH Ameliorates Renal Ischemia Reperfusion Injury via Activation of PI3K/AKT Signaling
Authors: C Hu, L Li, P Ding, L Li, X Ge, L Zheng, X Wang, J Wang, W Zhang, N Wang, H Gu, F Zhong, M Xu, R Rong, T Zhu, W Hu
J. Immunol., 2018-11-14;0(0):.
Species: Mouse
Sample Types: Serum
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Harmful Roles of TLR3 and TLR9 in Cardiac Dysfunction Developing during Polymicrobial Sepsis
Authors: F Fattahi, MW Russell, EA Malan, M Parlett, E Abe, FS Zetoune, PA Ward
Biomed Res Int, 2018-09-30;2018(0):4302726.
Species: Mouse
Sample Types: Plasma
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Complement depletion with cobra venom factor alleviates acute hepatic injury induced by ischemia?reperfusion
Authors: B Wang, H Xu, J Li, HM Gao, YH Xing, Z Lin, HJ Li, YQ Wang, SH Cao
Mol Med Rep, 2018-09-14;0(0):.
Species: Rat
Sample Types: Serum
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rIgG1 Fc Hexamer Inhibits Antibody-Mediated Autoimmune Disease via Effects on Complement and Fc?Rs
Authors: R Spirig, IK Campbell, S Koernig, CG Chen, BJB Lewis, R Butcher, I Muir, S Taylor, J Chia, D Leong, J Simmonds, P Scotney, P Schmidt, L Fabri, A Hofmann, M Jordi, MO Spycher, S Cattepoel, J Brasseit, C Panousis, T Rowe, DR Branch, A Baz Morell, F Käsermann, AW Zuercher
J. Immunol., 2018-03-12;0(0):.
Species: Mouse
Sample Types: Serum
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Chronic lung inflammation primes humoral immunity and augments antipneumococcal resistance
Authors: JD Boehme, S Stegemann-, A Autengrube, N Peters, J Wissing, L Jänsch, A Jeron, D Bruder
Sci Rep, 2017-07-10;7(1):4972.
Species: Mouse
Sample Types: BALF
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Attenuation of cGVHD by C5a/C5aR blockade is associated with increased frequency of Treg
Authors: Y Wang, P Lai, X Chen, C He, X Huang, S Geng, C Luo, S Wu, W Ling, L Zhong, Z Lu, P Li, J Weng, X Du
Sci Rep, 2017-06-15;7(1):3603.
Species: Mouse
Sample Types: Plasma
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Osteoblast-specific overexpression of complement receptor C5aR1 impairs fracture healing
Authors: S Bergdolt, A Kovtun, Y Hägele, A Liedert, T Schinke, M Amling, M Huber-Lang, A Ignatius
PLoS ONE, 2017-06-14;12(6):e0179512.
Species: Mouse
Sample Types: Serum
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TLR-Induced Murine Dendritic Cell (DC) Activation Requires DC-Intrinsic Complement
Authors: JH Sheen, MG Strainic, J Liu, W Zhang, Z Yi, ME Medof, PS Heeger
J. Immunol., 2017-05-24;0(0):.
Species: Mouse
Sample Types: Cell Culture Supernates
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Overexpression of Human CD55 and CD59 or Treatment with Human CD55 Protects against Renal Ischemia-Reperfusion Injury in Mice
Authors: AK Bongoni, B Lu, EJ Salvaris, V Roberts, D Fang, JL McRae, N Fisicaro, KM Dwyer, PJ Cowan
J. Immunol., 2017-05-12;0(0):.
Species: Mouse
Sample Types: Cell Culture Supernates
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Complement C5aR1 Signaling Promotes Polarization and Proliferation of Embryonic Neural Progenitor Cells through PKC?
Authors: LG Coulthard, OA Hawksworth, R Li, A Balachandr, JD Lee, F Sepehrband, N Kurniawan, A Jeanes, DG Simmons, E Wolvetang, TM Woodruff
J. Neurosci., 2017-04-28;0(0):.
Species: Mouse
Sample Types: CSF
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Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds
Authors: KM Cunnion, NK Krishna, HK Pallera, A Pineros-Fe, MG Rivera, PS Hair, BP Lassiter, R Huyck, MA Clements, AF Hood, GT Rodeheaver, PS Cottler, JL Nadler, AD Dobrian
PLoS ONE, 2017-01-20;12(1):e0170500.
Species: Mouse
Sample Types: Tissue Secretion
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High-fat diet-induced complement activation mediates intestinal inflammation and neoplasia, independent of obesity
Mol Cancer Res, 2016-08-17;0(0):.
Species: Mouse
Sample Types: Plasma
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The Complement System Component C5a Produces Thermal Hyperalgesia via Macrophage-to-Nociceptor Signaling That Requires NGF and TRPV1
J Neurosci, 2016-05-04;36(18):5055-70.
Species: Mouse
Sample Types: Tissue Homogenates
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Real-Time Imaging of Interactions of Neutrophils with Cryptococcus neoformans Demonstrates a Crucial Role of Complement C5a-C5aR Signaling.
Authors: Sun D, Zhang M, Liu G, Wu H, Zhu X, Zhou H, Shi M
Infect Immun, 2015-10-26;84(1):216-29.
Species: Mouse
Sample Types: Cell Culture Supernates
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The Complement Receptor C5aR Controls Acute Inflammation and Astrogliosis following Spinal Cord Injury.
Authors: Brennan F, Gordon R, Lao H, Biggins P, Taylor S, Franklin R, Woodruff T, Ruitenberg M
J Neurosci, 2015-04-22;35(16):6517-31.
Species: Mouse
Sample Types: Plasma
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Lysophosphatidic acid alters the expression profiles of angiogenic factors, cytokines, and chemokines in mouse liver sinusoidal endothelial cells.
Authors: Chou C, Lai S, Ho C, Lin W, Chen C, Lee P, Peng F, Kuo S, Wu S, Lai H
PLoS ONE, 2015-03-30;10(3):e0122060.
Species: Mouse
Sample Types: Cell Culture Supernates
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Complement component C5 recruits neutrophils in the absence of C3 during respiratory infection with modified vaccinia virus Ankara.
Authors: Price P, Banki Z, Scheideler A, Stoiber H, Verschoor A, Sutter G, Lehmann M
J Immunol, 2014-12-29;194(3):1164-8.
Species: Mouse
Sample Types: BALF
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The triggering receptor expressed on myeloid cells 2 inhibits complement component 1q effector mechanisms and exerts detrimental effects during pneumococcal pneumonia.
Authors: Sharif O, Gawish R, Warszawska J, Martins R, Lakovits K, Hladik A, Doninger B, Brunner J, Korosec A, Schwarzenbacher R, Berg T, Kralovics R, Colinge J, Mesteri I, Gilfillan S, Salmaggi A, Verschoor A, Colonna M, Knapp S
PLoS Pathog, 2014-06-12;10(6):e1004167.
Species: Mouse
Sample Types: BALF
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Complement C3 and C5 deficiency affects fracture healing.
Authors: Ehrnthaller, Christia, Huber-Lang, Markus, Nilsson, Per, Bindl, Ronny, Redeker, Simon, Recknagel, Stefan, Rapp, Anna, Mollnes, Tom, Amling, Michael, Gebhard, Florian, Ignatius, Anita
PLoS ONE, 2013-11-18;8(11):e81341.
Species: Mouse
Sample Types: Serum
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Deficiency of C5L2 increases macrophage infiltration and alters adipose tissue function in mice.
Authors: Gauvreau D, Gupta A, Fisette A, Tom F, Cianflone K
PLoS ONE, 2013-04-22;8(4):e60795.
Species: Mouse
Sample Types: Plasma
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Antigen-presenting cell-derived complement modulates graft-versus-host disease.
Authors: Kwan WH, Hashimoto D, Paz-Artal E
J. Clin. Invest., 2012-05-15;122(6):2234-8.
Species: Mouse
Sample Types: Cell Culture Supernates
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Increased local concentration of complement C5a contributes to incisional pain in mice.
Authors: Jang JH, Liang D, Kido K
J Neuroinflammation, 2011-07-07;8(0):80.
Species: Mouse
Sample Types: Tissue Homogenates
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Complement regulates CD4 T-cell help to CD8 T cells required for murine allograft rejection.
Authors: Vieyra M, Leisman S, Raedler H, Kwan WH, Yang M, Strainic MG, Medof ME, Heeger PS
Am. J. Pathol., 2011-06-23;179(2):766-74.
Species: Mouse
Sample Types: Cell Culture Supernates
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The C5a Receptor on Mast Cells Is Critical for the Autoimmune Skin-blistering Disease Bullous Pemphigoid.
Authors: Heimbach L, Li Z, Berkowitz P
J. Biol. Chem., 2011-03-10;286(17):15003-9.
Species: Mouse
Sample Types: Tissue Homogenates
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A protective role for complement C3 protein during pandemic 2009 H1N1 and H5N1 influenza A virus infection.
Authors: O'Brien KB, Morrison TE, Dundore DY
PLoS ONE, 2011-03-09;6(3):e17377.
Species: Mouse
Sample Types: BALF
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Distinct Different Contributions of the Alternative and Classical Complement Activation Pathway for the Innate Host Response during Sepsis.
Authors: Dahlke K, Wrann CD, Sommerfeld O, Sossdorf M, Recknagel P, Sachse S, Winter SW, Klos A, Stahl GL, Ma YX, Claus RA, Reinhart K, Bauer M, Riedemann NC
J. Immunol., 2011-01-24;186(5):3066-3075.
Species: Mouse
Sample Types: Plasma
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The absence of serum IgM enhances the susceptibility of mice to pulmonary challenge with Cryptococcus neoformans.
Authors: Subramaniam KS, Datta K, Quintero E, Manix C, Marks MS, Pirofski LA
J. Immunol., 2010-04-19;184(10):5755-67.
Species: Mouse
Sample Types: Tissue Homogenates
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