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Aging/Glycation Assay Kit series

Collagen AGEs Assay Kits


Collagen AGEs Assay Kit [PDF]

 back to "Advanced Glycation End Products (AGEs) Research" page

Background

Although carbohydrates are indispensable for ATP production, excess amounts of carbohydrates modify amino residues of amino acids such as lysine and arginine, and results in the irreversible functional disorders of proteins by changing the three-dimensional structure and net negative charge in patients with disordered metabolism. Since this reaction was first reported by Louis Camille Maillard in 1912, the reaction is called the Maillard reaction, or glycation.

The Maillard reaction is divided by early and advanced stages. Early stage generates Amadori rearrangement products, such as haemoglobin A1c, whereas advanced stage generates the AGEs (advanced glycation end products), which is characterized by colour in brown and protein cross-linking. Collagen, the structural protein that forms skin, blood vessel wall and bone, also undergo glycation reaction.

CML is generated by the oxidative cleavage of Amadori products by hydroxyl radicals, peroxynitrite  and hypochloric acid, thus suggesting that CML is an important biological marker of oxidative stress in vivo. CML concentration, adjusted for age and duration of diabetes, is further increased in patients who have severe complications, including nephropathy, retinopathy, and atherosclerosis.

Nε-carboxymethylarginine (CMA), an AGE component was identified in glycated collagen, it generates during the reaction of collagen with reducing sugars or glyoxal. AGEs accumulation in collagen induced dermal fibroblasts to undergo apoptosis. Because AGEs accumulate in collagen as a function of aging, CMA may be involved in aging of collagen-rich tissues such as skin. CML is detected in many proteins such as collagen and albumin, whereas CMA is generates specifically in collagen, suggesting that CMA may provide a marker for collagen glycation. An anti-CMA monoclonal antibody specifically and sensitively detects CMA in collagen.

CML ELISA Assay Kit provides rapid detection of CML formed by glycation with glyoxal on the collagen coating plate.

CMA ELISA Assay Kit provides rapid detection of CMA formed by glycation with glyoxal on the collagen coating plate.

These kits are suitable to the research for functional foods and cosmetic materials which have anti-glycation activity.

 

Principle

Collagen coated on 96-well plate is glycated by glyoxal and CML or CMA is formed onto collagen coating wells for 7days at 37 °C. The inhibitory effects of glycation by aminoguanidin (positive control) or samples are probed with an anti-CML or CMA antibody, followed by an HRP conjugated secondary antibody. The inhibitory effect of glycation in unknown sample is determined by comparing with the inhibitory effect of glycation by aminoguanidin (positive control). “Collagen AGEs Assay Kit, CML or CMA-Specific, Glyoxal” is suitable to the research for functional foods and cosmetic materials which have an anti-glycation activity.

Kit component

Collagen AGEs Assay Kit, CML-Specific, Glyoxal

ComponentContentStorage
96-well Collagen coated plate One strip-well plate 4C
Microplate seal 2 Sheets 4C
Anti-CML Antibody(100X)  (Cat.# CSR-AAS-AGE-K02E)
          or
Anti-CMA Antibody(100X)  (Cat.# CSR-AAS-AGE-K03E)
100 uL 4C
Blocking Buffer 50 mL 4C
HRP Conjugate Secondary Antibody(100X) 100 uL 4C
Sample Dilution Buffer 30 mL 4C
Washing Buffer(10X) 50 mL 4C
Substrate Solution 10 uL 4C
Stop Solution 10 uL 4C
Glyoxal Solution 5 mL 4C
Aminoguanidin Solution(10 mM) ※ positive control Aminoguanidin Solution(10 mM) *positive control 250 uL 4C

Example Experiments


Inhibitory effect of aminoguanidine and phytochemicals on the CML formation.


Inhibitory effect of aminoguanidine and phytochemicals on the CMA formation.

Product List

Product Name Cat# Quantity Price

Collagen AGEs Assay Kit, CML-Specific, Glyoxal

CSR-AAS-AGE-K02E 1KIT

¥ 80,000
$ 1067
€ 800

Collagen AGEs Assay Kit, CMA-Specific, Glyoxal

CSR-AAS-AGE-K03E 1KIT

¥ 80,000
$ 1067
€ 800

References

1. Iijima K, Murata M, Takahara H, et al: Identification of Nω -carboxymethylarginine as a novel acid-labile advanced glycation end product in collagen. J Biochem 347; 23?27: 2000

2. Alikhani Z, Alikhani M, Boyd CM, et al: Advanced Glycation End Products Enhance Expression of Pro-apoptotic Genes and Stimulate Fibroblast Apoptosis through Cytoplasmic and Mitochondrial Pathways. J Biol Chem 280; 12087?12095: 2005

3. Verzijl N, DeGroot J, Oldehinkel E, Bank RA, Thorpe SR, Baynes JW, Bayliss MT, Bijlsma JW, Lafeber FP, Tekoppele JM. Age-related accumulation of Maillard reaction products in human articular cartilage collagen. Biochem J. 2000 Sep 1;350 Pt 2:381-387

4. Shimasaki S, Kubota M, Yoshitomi M, Takagi K, Suda K, Mera K, Fujiwara Y, Nagai R. Nω -(carboxymethyl)arginine Accumulates in Glycated Collagen and Klotho-deficient Mouse Skin. Anti-Aging Medicine 8 (6) : 82-87, 2011

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.