Human Serum Amyloid A1 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 Serum Amyloid A1. The suggested diluent is suitable for the analysis of most cell culture supernate, serum, and plasma 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 3 (5 plates): (Catalog # DY009) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 3.
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 # DY004), or 5% Tween 20 in PBS, 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: Serum Amyloid A1
The apolipoproteins are a structurally-unrelated group of proteins that have some association with the transport of lipids in blood. Apolipoproteins, plus phospholipids, cholesterol and triglycerides, form spherical particles with a lipid/hydrophobic center and a (apolipo)protein coat. The apolipoprotein coat promotes aqueous solubility and serves as a ligand for lipoprotein receptors. HDL may contain apolipoproteins A, C, D, E, J, L and M, while LDL contains apolipoproteins B and E.
ApoAI and ApoA2 are major protein components of serum high-density lipoprotein (HDL) and are produced by the liver and small intestine. They are involved in reverse cholesterol transport from tissues to the liver. Polymorphisms of ApoA2 are associated with disorders of cholesterol and fatty acid metabolism. Human ApoB (Apolipoprotein B-100) is a 550 kDa, secreted, palmitoylated glycoprotein that is part of LDL and VLDL particles. It is made by liver and is 4536 aa in length. It binds LDL to the ApoB/E receptor. ApoC activates lipoprotein lipase and may self-associate to form amyloid-type fibrils.
ApoE is a 34 kDa protein component of serum chylomicrons, VLDL, and HDL particles. It mediates the binding, uptake, and catabolism of these particles through interactions with the ApoE receptor and LDL receptors in the liver and brain. ApoE is important in fatty acid homeostasis and memory formation. Polymorphisms encode three variants (ApoE2, 3, 4) which are differentially related to the development of atherosclerosis and neurogenerative disorders, particularly Alzheimer's disease.
Serum amyloid A proteins (SAAs) are a family of homologous apolipoproteins of high density lipoprotein (HDL). They can be divided into two groups. The first group consists of the acute phase SAA1 and SAA2 that associate with HDL during inflammation and remodel the HDL particle by displacing apolipoprotein A1. The second group consists of constitutively expressed SAA4 and SAA5 that exist as minor apolipoproteins on HDL but make up more than 90% of the total SAA during homeostasis.
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 of 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 Serum Amyloid A1 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.
15
Citations: Showing 1 - 10
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Favourable HDL composition in endurance athletes is not associated with changes in HDL in vitro antioxidant and endothelial anti-inflammatory function
Authors: Beazer, JD;Sillars, A;Beck, S;Christoffersen, C;Ferraz, MJ;Mulder, MT;Graham, D;Karlsson, H;Ljunggren, S;Gill, J;Freeman, DJ;
Bioscience reports
Species: Human
Sample Types: Serum
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Lactate-to-albumin ratio and cholesterol levels predict neurological outcome in cardiac arrest survivors
Authors: Chen, DL;Chung, CM;Wang, GJ;Chang, KC;
The American journal of emergency medicine
Species: Human
Sample Types: Plasma
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Hepcidin as a Diagnostic Biomarker in Anaemic Lung Cancer Patients
Authors: K Wadowska, P B?asiak, A Rzechonek, I Bil-Lula, M ?liwi?ska-
Cancers, 2022-12-30;15(1):.
Species: Human
Sample Types: Serum
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Serum protein profiling of lung, pancreatic, and colorectal cancers reveals alcohol consumption-mediated disruptions in early-stage cancer detection
Authors: H Lee, SY Kim, DW Kim, YS Park, JH Hwang, S Cho, JY Cho
Heliyon, 2022-12-15;8(12):e12359.
Species: Human
Sample Types: Serum
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Effect of N-3 Polyunsaturated Fatty Acids on Lipid Composition in Familial Hypercholesterolemia: A Randomized Crossover Trial
Authors: LN Hande, C Kjellmo, K Pettersen, S Ljunggren, H Karlsson, K Cederbrant, M Marcusson-, A Hovland, KT Lappegård
Biomedicines, 2022-07-27;10(8):.
Species: Human
Sample Types: Plasma
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Oxidative stress and inflammation parameters-novel biomarkers for idiopathic pulmonary fibrosis
Authors: M Roksandic, A Klisic, V Ceriman, J Kotur Stev, K Savic Vujo, D Mirkov, M Gajic, B Ilic, N Dimic, N Samardzic, D Jovanovic
European review for medical and pharmacological sciences, 2022-02-01;26(3):927-934.
Species: Human
Sample Types: Plasma
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New Insights on Old Biomarkers Involved in Tumor Microenvironment Changes and Their Diagnostic Relevance in Non-Small Cell Lung Carcinoma
Authors: K Wadowska, P B?asiak, A Rzechonek, I Bil-Lula, M ?liwi?ska-
Biomolecules, 2021-08-13;11(8):.
Species: Human
Sample Types: Serum
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Metal additive manufacturing and possible clinical markers for the monitoring of exposure-related health effects
Authors: SA Ljunggren, LJ Ward, P Graff, A Persson, ML Lind, H Karlsson
PLoS ONE, 2021-03-18;16(3):e0248601.
Species: Human
Sample Types: Plasma
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Subclinical effects of remote ischaemic conditioning in human kidney transplants revealed by quantitative proteomics
Authors: AM Thorne, H Huang, DP O'Brien, M Eijken, NV Krogstrup, R Norregaard, B Møller, RJ Ploeg, B Jespersen, BM Kessler
Clinical Proteomics, 2020-11-02;17(1):39.
Species: Human
Sample Types: Protein
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Serum amyloid A is a soluble pattern recognition receptor that drives type 2 immunity
Authors: U Smole, N Gour, J Phelan, G Hofer, C Köhler, B Kratzer, PA Tauber, X Xiao, N Yao, J Dvorak, L Caraballo, L Puerta, S Rosskopf, J Chakir, E Malle, AP Lane, WF Pickl, S Lajoie, M Wills-Karp
Nat. Immunol., 2020-06-22;21(7):756-765.
Species: Human
Sample Types: Serum
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Expression of SAA1, SAA2 and SAA4 genes in human primary monocytes and monocyte-derived macrophages
Authors: C Jumeau, F Awad, E Assrawi, L Cobret, P Duquesnoy, I Giurgea, D Valeyre, G Grateau, S Amselem, JF Bernaudin, SA Karabina
PLoS ONE, 2019-05-17;14(5):e0217005.
Species: Human
Sample Types: Cell Culture Supernates
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Modified lipoproteins in periodontitis: a link to cardiovascular disease?
Authors: S Ljunggren, T Bengtsson, H Karlsson, C Starkhamma, E Palm, F Nayeri, B Ghafouri, J Davies, G Svensäter, J Lönn
Biosci. Rep., 2019-03-26;39(3):.
Species: Human
Sample Types: Plasma
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Bacterial Lipoproteins Constitute the TLR2-Stimulating Activity of Serum Amyloid A
Authors: EJ Burgess, LR Hoyt, MJ Randall, MM Mank, JJ Bivona, PL Eisenhauer, JW Botten, BA Ballif, YW Lam, MJ Wargo, JE Boyson, JL Ather, ME Poynter
J. Immunol., 2018-08-29;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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Induction of pro-inflammatory genes by serum amyloid A1 in human amnion fibroblasts
Authors: W Li, W Wang, R Zuo, C Liu, Q Shu, H Ying, K Sun
Sci Rep, 2017-04-06;7(1):693.
Species: Human
Sample Types: Cell Culture Supernates
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Alterations in high-density lipoprotein proteome and function associated with persistent organic pollutants
Authors: Mats Lindahl
Environ Int, 2016-11-16;98(0):204-211.
Species: Human
Sample Types: High Density Lipoprotein
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Average Rating: 4.7 (Based on 6 Reviews)
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This assay works very well with some assay optimization
We used this kit for the quantification of SAA1 in human serum and plasma. Works very well and well-described protocol.
