Human Apolipoprotein A-I/ApoA1 DuoSet ELISA

Catalog # Availability Size / Price Qty
DY3664-05
Ancillary Products Available
Human Apolipoprotein A-I / ApoA1 ELISA Standard Curve
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Product Details
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Human Apolipoprotein A-I/ApoA1 DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sufficient Materials
For five 96-well plates*
Specificity
Please see the product datasheet

* 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 Apolipoprotein A-1 (ApoA1). 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 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)

Data Example

Human Apolipoprotein A-I / ApoA1 ELISA Standard Curve

Product Datasheets

Preparation and Storage

Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: Apolipoprotein A-I/ApoA1

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.

Entrez Gene IDs:
335 (Human); 11806 (Mouse); 25081 (Rat)
Alternate Names:
Alp-1; APOA1; Apo-AI; ApoA-I; Apolipoprotein A1; Apolipoprotein AI; Apolipoprotein A-I; Brp-14; Ltw-1; Lvtw-1; MGC117399; Sep-1; Sep-2

Assay Procedure

GENERAL ELISA PROTOCOL

Plate Preparation

  1. 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.
  2. 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.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. 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.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. 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.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. 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.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. 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.

FAQs

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