OxLDL ELISA Kit

Catalog No. ABIN2345041

Product Details OxLDL ELISA Kit

Target: OxLDL (Oxidized Low Density Lipoprotein (OxLDL))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Brand: OxiSelect™

Sample Type: Serum, Plasma

Analytical Method: Quantitative

Sensitivity: 15 ng/mL

Characteristics: The OxiSelect™ Human Oxidized LDL ELISA Kit is an enzyme immunoassay developed for the detection and quantitation of human OxLDL in plasma, serum or other biological fluid samples. The kit contains an OxLDL Standard and has a detection sensitivity limit of <15 ng/mL. Each kit provides sufficient reagents to perform up to 96 assays including standard curve and unknown samples.

Components:

1. Anti-MDA Antibody Coated Plate : One 96-well strip plate.
2. Biotinylated Anti-Human ApoB-100 Antibody (1000X) : One 20 μL vial.
3. LDL Precipitation Solution (2X) : One 20 mL bottle.
4. Streptavidin-Enzyme Conjugate : One 20 μL vial.
5. Assay Diluent : One 50 mL bottle.
6. 10X Wash Buffer : One 100 mL bottle.
7. Substrate Solution : One 12 mL amber bottle.
8. Stop Solution (Part. No. 310808): One 12 mL bottle.

Box 2 (shipped on blue ice packs)

Material not included:

1. Human Plasma or Serum Samples
2. PBS
3. Microcentrifuge
4. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
5. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
6. Multichannel micropipette reservoir 4
7. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Suitable for use with human plasma or serum samples
• Copper oxidized LDL (oxLDL) Standard included
• Detection sensitivity limit of 50 ng/mL for MDA-LDL

Plate: Pre-coated

Reagent Preparation:

• 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.
• Blocking Reagent: Immediately before use dilute the Blocking Reagent 1:100 with PBS. Do not store diluted solutions.
• Biotinylated Anti-Human ApoB-100 Antibody and Streptavidin-Enzyme Conjugate: Immediately before use dilute the Anti-ApoB-100 antibody 1:1000 and Streptavidin-Enzyme Conjugate 1:1000 with Assay Diluent. Do not store diluted solutions.

Sample Preparation:

The following recommendations are only guidelines and may be altered to optimize or complement the user's experimental design.

• Plasma: Collect blood with heparin or EDTA and centrifuge for 10 minutes at 1000 x g at 4 °C. Remove 200 μL of plasma and add 200 μL of LDL Precipitation Solution, mixing well. Incubate at room temperature for 5 minutes (precipitation will occur). Centrifuge for 20 minutes at 2000 x g (pellet should be visible). Carefully aspirate the supernatant and collect the pellet. Resuspend and dissolve the pellet in 1.6 mL of PBS, vortexing well. Further dilute the sample 1:50 to 1:200 in Assay Diluent before running the ELISA. Assay immediately and do not store solutions.
• Serum: Harvest serum and centrifuge for 10 minutes at 1000 x g at 4 °C. Remove 200 μL of serum and add 200 μL of LDL Precipitation Solution, mixing well. Incubate at room temperature for 5 5 minutes (precipitation will occur). Centrifuge for 20 minutes at 2000 x g (pellet should be visible). Carefully aspirate the supernatant and collect the pellet. Resuspend and dissolve the pellet in 1.6 mL of PBS, vortexing well. Further dilute the sample 1:50 to 1:200 in Assay Diluent before running the ELISA. Assay immediately and do not store solutions.

Assay Procedure:

1. For plasma and serum samples, refer to the above Sample Preparation Section. These samples require LDL Precipitation Solution treatment immediately prior to running the assay.
2. Add 100 μL of OxLDL standard or unknown sample to the Anti-MDA Antibody Coated Plate. Each OxLDL standard, blank and unknown sample should be assayed in duplicate.
3. Cover with a plate cover and incubate at room temperature for 2 hours on an orbital shaker.
4. Wash microwell strips 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
5. Add 100 μL of diluted Blocking Reagent to each well. Cover with a plate cover and incubate at room temperature for 1 hour on an orbital shaker.
6. Wash microwell strips 5 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
7. Add 100 μL of the diluted Biotinylated Anti-Human ApoB-100 antibody to each well. Incubate at room temperature for 1 hour on an orbital shaker.
8. Wash the strip wells 5 times according to step 6 above.
9. Add 100 μL of the diluted Streptavidin-Enzyme Conjugate to each well. Incubate at room temperature for 1 hour on an orbital shaker.
10. Wash the strip wells 5 times according to step 6 above. Proceed immediately to the next step.
11. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation time may vary from 5-20 minutes. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation. 6
12. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).
13. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length.

Restrictions: For Research Use only

Handling

Handling Advice: Avoid multiple freeze/thaw cycles.

Storage: 4 °C/-20 °C

Storage Comment: Upon receipt, aliquot and store the Blocking Reagent at -20°C to avoid multiple freeze/thaw cycles. Store all other components at 4°C.

References for OxLDL ELISA Kit (ABIN2345041)

Wang, Liu, Cong, Li, Li, Yan, Tang, Cheng, Zheng et al.: "Shift of the interconnection from the reaction system of paraoxonase 1 to the peroxidation reaction system of myeloperoxidase with HDL-C levels: a marker of atherosclerosis in patients with normal ..." in: Clinica chimica acta; international journal of clinical chemistry, Vol. 438, pp. 370-5, (2014) (PubMed).

Stojanov, Stefanovic, Dzingalasevic, Ivanisevic, Miljkovic, Mandic-Radic, Prostran: "Total bilirubin in young men and women: association with risk markers for cardiovascular diseases." in: Clinical biochemistry, Vol. 46, Issue 15, pp. 1516-9, (2013) (PubMed).

Target Details for OxLDL

Target: OxLDL (Oxidized Low Density Lipoprotein (OxLDL))

Abstract: OxLDL Products

Background: Lipoproteins are submicroscopic particles composed of lipid and protein held together by noncovalent forces. Their general structure is that of a putative spheroidal microemulsion formed from an outer layer of phospholipids, unesterified cholesterol, and proteins, with a core of neutral lipids, predominately cholesteryl esters and triacylglycerols (TAG). Low density lipoprotein (LDL) is the major transport protein for cholesterol in human plasma. LDL is a spherical particle with a diameter of 20-25 nm. Each LDL particle contains cholesteryl esters in its core which are surrounded by a hydrophilic coat composed of phospholipids, cholesterol, and one molecule of a hydrophobic protein known as apolipoprotein B-100 (Figure 1). Figure 1: Structure of LDL. LDL cholesterol, sometimes referred to as "bad" cholesterol, is even more dangerous when it becomes oxidized. Oxidized LDL (OxLDL) is more reactive with surrounding tissues and can collect within the inner-lining of arteries. Macrophages, cholesterol, and other lipids can accumulate at the site (atherosclerosis), ultimately forming a plaque that can lead to heart attack, stroke or death. LDL oxidation affects both the lipid and protein components of LDL. Reactive aldehyde products formed during the oxidation of polyunsaturated fatty acids, such as malondialdehyde (MDA) and 4- hydroxynonenal (HNE), are capable of attaching covalently to the ε-amino groups of lysine residues of of ApoB-100 to form MDA-Lys and HNE-Lys adducts (MDA-LDL and HNE-LDL). Advanced glycosylation, such as the formation of CML-LDL and CEL-LDL, are also involved in LDL oxidation.