Human APP 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 APP (Amyloid Precursor Protein). 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: APP
Amyloid Precursor Protein (APP) is a type I transmembrane protein that is ubiquitously expressed on cell surfaces. It undergoes complex proteolytic processing and is cleaved by alpha-, beta-, and gamma-Secretases to generate soluble APP alpha, soluble APP beta, and Amyloid beta (A beta) fragments of several lengths. One of these fragments, A beta 42, generated by beta- and gamma-Secretase activities, has been implicated in Alzheimer's disease. Aberrantly high levels of this peptide form and accumulate in the brains of Alzheimer's disease patients to create the senile plaques characteristic of the disease.
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 APP 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 - 5
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Cerebrospinal fluid NCAM-1 concentration is associated with neurodevelopmental outcome in post-hemorrhagic hydrocephalus of prematurity
Authors: DD Limbrick, DM Morales, CN Shannon, JC Wellons, AV Kulkarni, JS Alvey, RW Reeder, V Freimann, R Holubkov, JK Riva-Cambr, WE Whitehead, CJ Rozzelle, M Tamber, WJ Oakes, JM Drake, IF Pollack, RP Naftel, TE Inder, JR Kestle, Hydrocepha
PLoS ONE, 2021-03-10;16(3):e0247749.
Species: Human
Sample Types: CSF
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Plasma biomarkers of the amyloid pathway are associated with geographic atrophy secondary to age-related macular degeneration
Authors: K Lashkari, GC Teague, U Beattie, J Betts, S Kumar, MM McLaughlin, FJ López
PLoS ONE, 2020-08-07;15(8):e0236283.
Species: Human
Sample Types: Plasma
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Cerebrospinal fluid biomarkers of infantile congenital hydrocephalus
Authors: DD Limbrick, B Baksh, CD Morgan, G Habiyaremy, JP McAllister, TE Inder, D Mercer, DM Holtzman, J Strahle, MJ Wallendorf, DM Morales
PLoS ONE, 2017-02-17;12(2):e0172353.
Species: Human
Sample Types: CSF
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Cerebrospinal fluid levels of amyloid precursor protein are associated with ventricular size in post-hemorrhagic hydrocephalus of prematurity.
Authors: Morales, Diego M, Holubkov, Richard, Inder, Terri E, Ahn, Haejun C, Mercer, Deanna, Rao, Rakesh, McAllister, James P, Holtzman, David M, Limbrick, David D
PLoS ONE, 2015-03-04;10(3):e0115045.
Species: Human
Sample Types: CSF
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Evidence of a novel mechanism for partial gamma-secretase inhibition induced paradoxical increase in secreted amyloid beta protein.
Authors: Barnwell E, Padmaraju V, Baranello R, Pacheco-Quinto J, Crosson C, Ablonczy Z, Eckman E, Eckman C, Ramakrishnan V, Greig N, Pappolla M, Sambamurti K
PLoS ONE, 2014-03-21;9(3):e91531.
Species: Human
Sample Types: Cell Culture Supernates
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