AGE ELISA Kit (Advanced Glycation End Product)

Details for Product AGE ELISA Kit No. ABIN2345151, Supplier: Log in to see
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Method Type
Competition ELISA
Application
ELISA
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Purpose First, an AGE conjugate is coated on an ELISA plate. The unknown AGE protein samples or AGE- BSA standards are then added to the AGE conjugate preabsorbed ELISA plate. After a brief incubation, an anti-AGE polyclonal antibody is added, followed by an HRP conjugated secondary antibody. The content of AGE protein adducts in unknown samples is determined by comparison with a predetermined AGE-BSA standard curve.
Brand OxiSelect™
Sample Type Cell Lysate, Purified Protein, Serum, Plasma
Analytical Method Quantitative
Detection Method Colorimetric
Sensitivity 0.5 μg/mL
Characteristics OxiSelect™ AGE Competitive ELISA Kit is an enzyme immunoassay developed for rapid detection and quantitation of AGE protein adducts. The quantity of AGE adduct in protein samples is determined by comparing its absorbance with that of a known AGE-BSA standard curve. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown protein samples.
Components
  1. 96-well Protein Binding Plate : One strip well 96-well plate.
  2. Anti-AGE Antibody (1000X) : One 10 μL vial of anti-AGE antibody.
  3. Secondary Antibody, HRP Conjugate (1000X) : One 20 μL vial.
  4. Assay Diluent : One 50 mL bottle.
  5. 10X Wash Buffer : One 100 mL bottle.
  6. Substrate Solution : One 12 mL amber bottle.
  7. Stop Solution (Part. No. 310808): One 12 mL bottle.

Box 2 (shipped on blue ice packs)

Material not included
  1. Protein samples such as purified protein, plasma, serum, cell lysate
  2. 1X PBS
  3. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
  4. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
  5. Multichannel micropipette reservoir
  6. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)
Background The non-enzymatic reaction of reducing carbohydrates with lysine side chains and N-terminal amino groups of macromolecules (proteins, phospholipids and nucleic acids) is called the Maillard reaction or glycation. The products of this process, termed advanced glycation end products (AGEs), adversely affect the functional properties of proteins, lipids and DNA. For example, N-ε-(Carboxymethyl) lysine (CML), one of the prevalent AGEs, has been implicated in oxidative stress and vascular damage. Tissue levels of AGE increase with age and the formation of AGEs is predominantly endogenous, though these products can also be derived from exogenous sources such as food and tobacco smoke. AGE modification of proteins can contribute to the pathophysiology of aging and long-term complications of diabetes, atherosclerosis and renal failure. AGEs also interact with a variety of cell- surface AGE-binding receptors (RAGE), leading either to their endocytosis and degradation or to cellular activation and pro-oxidant or pro-inflammatory events.
Comment

  • Detect advanced glycation end product formation as low as 0.5 μg/mL
  • AGE-modified BSA and Reduced BSA included as standards
  • Compatible with cell lysates, plasma, serum, and purified proteins

Plate Uncoated
Reagent Preparation
  • AGE Conjugate Coated Plate: Note: The AGE Conjugate coated wells are not stable and should be used within 24 hrs after coating. Only coat the number of wells to be used immediately. 1. Immediately before use, prepare 1X Conjugate Diluent by diluting the 100X Conjugate Diluent in 1X PBS. Example: Add 50 μL to 4.95 mL of 1X PBS. 2. Immediately before use, prepare 10 μg/mL of AGE Conjugate by diluting the 1.0 mg/mL AGE Conjugate in 1X PBS. Example: Add 25 μL to 2.475 mL of 1X PBS. 3 3. Mix 10 μg/mL of AGE Conjugate and 1X Conjugate Diluent at 1:1 ratio and add 100 μL of the mixture to each well and incubate overnight at 4 °C. Remove the AGE Conjugate coating solution and wash twice with 1X PBS. Blot plate on paper towels to remove excess fluid. Add 200 μL of Assay Diluent to each well and block for 1 hr at room temperature. Transfer the plate to 4 °C and remove the Assay Diluent immediately before use.
  • 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.
  • Anti-AGE Antibody and Secondary Antibody: Immediately before use, dilute the Anti-AGE antibody 1:1000 and Secondary Antibody 1:1000 with Assay Diluent. Do not store diluted solutions.
Assay Procedure
  1. Prepare and mix all reagents thoroughly before use. Each AGE sample including unknown and standard should be assayed in duplicate.
  2. Add 50 μL of unknown sample or AGE-BSA standard to the wells of the AGE Conjugate coated plate. If needed, unknown samples may be diluted in 1X PBS containing 0.1 % BSA before adding. Incubate at room temperature for 10 minutes on an orbital shaker.
  3. Add 50 μL of the diluted anti-AGE antibody to each well, incubate at room temperature for 1 hour on an orbital shaker. 4
  4. Wash 3 times with 250 μL of 1X Wash Buffer 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 the diluted Secondary Antibody-HRP Conjugate to all wells and incubate for 1 hour at room temperature on an orbital shaker. Wash the strip wells 3 times according to step 4 above.
  6. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well. Incubate at room temperature for 2-20 minutes on an orbital shaker. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation.
  7. Stop the enzyme reaction by adding 100 μL of Stop Solution to each well. Results should be read immediately (color will fade over time).
  8. Read absorbance of each well on a microplate reader using 450 nm as the primary wave length. 5
Restrictions For Research Use only
Handling Advice Avoid multiple freeze/thaw cycles.
Storage 4 °C/-20 °C
Storage Comment Upon receipt, aliquot and store the Anti-AGE Antibody, AGE-BSA Standard, AGE Conjugate and 100X Conjugate Diluent at -20°C to avoid multiple freeze/thaw cycles. Store all other kit components at 4°C.
Product cited in: Chen, Chen, Wu, Chou, Chen, Wei, Lee, Hong, Zheng, Chiu, Lin, Hsu, Hsu: "Far-infrared protects vascular endothelial cells from advanced glycation end products-induced injury via PLZF-mediated autophagy in diabetic mice." in: Scientific reports, Vol. 7, pp. 40442, 2017 (PubMed).

Martins, Oliveira, Vizcaíno, Fonseca, Gouveia, Silva, Henriques, Noronha, Rodrigues: "Advanced Glycation End Products Evolution after Pancreas-Kidney Transplantation: Plasmatic and Cutaneous Assessments." in: Oxidative medicine and cellular longevity, Vol. 2016, pp. 2189582, 2016 (PubMed).

Richarme, Marguet, Forterre, Ishino, Ishino: "DJ-1 family Maillard deglycases prevent acrylamide formation." in: Biochemical and biophysical research communications, Vol. 478, Issue 3, pp. 1111-6, 2016 (PubMed).

Caram, Ferrari, Bertani, Garcia, Mesquita, Knaut, Tanni, Godoy: "Smoking and Early COPD as Independent Predictors of Body Composition, Exercise Capacity, and Health Status." in: PLoS ONE, Vol. 11, Issue 10, pp. e0164290, 2016 (PubMed).

Chao, Chen, Chang, Lee, Wang, Chang: "Effects of ferric citrate supplementation on advanced glycation end products in a rat model of streptozotocin/nicotinamide-induced diabetes." in: Molecular nutrition & food research, Vol. 61, Issue 5, 2016 (PubMed).

Chen, Aikawa, Yoshida, Matsui: "Methylglyoxal-derived hydroimidazolone residue of plasma protein can behave as a predictor of prediabetes in Spontaneously Diabetic Torii rats." in: Physiological reports, Vol. 3, Issue 8, 2015 (PubMed).

Foster, Spruill, Walter, Nogueira, Fedarovich, Turner, Ahmed, Salley, Ford, Findlay, Turner: "AGE metabolites: a biomarker linked to cancer disparity?" in: Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, Vol. 23, Issue 10, pp. 2186-91, 2014 (PubMed).

de Laat, Kyaw-Tanner, Sillence, McGowan, Pollitt: "Advanced glycation endproducts in horses with insulin-induced laminitis." in: Veterinary immunology and immunopathology, Vol. 145, Issue 1-2, pp. 395-401, 2012 (PubMed).