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Accurate measurement of native type II collagen from tissue or cell culture

ELISA Assay Kit To Measure Native Type II Collagen


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Background

There are more than 20 known types of collagen and they are classified by a Roman numeral based on their time of discovery. They are found in all connective tissues such as skin, bone, cartilage, vasculature, tendons and ligaments where their function is to provide support. All collagen molecules consist of three polypeptide chains folded into a left-handed helical conformation, and the three helical chains are then wrapped around each other into a right-handed triple helix. Homotrimer collagen types have 3 identical chains, whereas heterotimer collagen types contain two or three different chains. Everythird amino acid is glycine in the repeating Gly-X-Y sequence in each of the chains. Glycine allows for the triple helix confirmation where the three chains come together and X is usually proline and Y, hydroxyproline.

Types I, II, III, V and XI are considered the fibril-forming collagens as they form long fibers consisting of a triple-helical domain and are similar in structure. They are characterized by 67 nm banded fibrils that are quarter -staggered. The length of these collagens is approximately 300 nm and 1.5nm in diameter and consist of 1000 Gly-X-Y repeating residues. Collagens are synthesized as precursor molecules called procollagens that contain amino (N) and carboxy (C) terminal propeptide domains which consist of collagen and non-collagen-like sequences. The N and C terminal propeptides are linked to the main triple-helical domain by short, non-collagenous sequences, called telopeptides. The telopeptides are the primary sites for intermolecular cross-linking, which is important for the stabilization of the collagen fibers.

The procollagen molecules are processed to mature collagen molecules by cleavage of the N and C propeptides by specific N and C proteinases.

Type II collagen is homotrimeric consisting of three identical alpha1(II) chains and is the major collagen of cartilage accounting for approximately 95% of all collagen types. Type II collagen is also in the vitreous body of the eye, the nucleus pulposus of intervertebral discs and the inner ear. The COL2A1 gene codes for the production of the pro- alpha1(II) chain of type II collagen. It has been discovered that the gene sequence coding for the N-propeptide has an alternatively spliced exon that codes for an additional 69-amino acid cysteine-rich domain. Type IIA procollagen contains this cysteine-rich globular domain while type IIB lacks it and both have distinct distributions during various stages of chondrogenesis. Type IIA procollagen can be localized to pre-chondrocytes of the perichondrium, while type IIB is localized to differentiated chondrocytes of cartilage.

Autoimmunity to type II collagen is implicated in the pathogenesis of various diseases including rheumatoid arthritis (RA), eye disease associated with RA, and relapsing polychondritis. Circulating antibodies to type II collagen are found in these diseases and rheumatoid cartilage and synovium contain antibodies to type II collagen more than in serum. Furthermore, immunization of susceptible rodents and non-human primates with native, type II collagen (collagen-induced arthritis; CIA) induces an erosive polyarthritis which closely resembles RA. In CIA, complement fixing antibodies bind to type II collagen in autologous cartilage, and initiate the inflammation cascade. The CIA model has proven to be a valuable model to study the pathogenesis of arthritis and for the development of new therapies.

In cartilage, type II collagen is polymerized to form collagen fibrils. Small-diameter fibrils (10-25 nm) are formed pericellularly, while larger fibrils (up to 300 nm in diameter) are formed in the territorial and interterritorial matrix. The collagen molecules in the fibrils overlap by a quarter-stagger forming a banded pattern. The molecules are covalently cross-linked between the triple-helical domains and the telopeptides.

The Rheumera™ kit is specific for measuring native type II collagen, and will not measure denatured collagen. This kit will measure 80 samples, individually or 40 samples in duplicate, and the sensitivity of this assay is in the nanogram range. The Native Type II Collagen Detection Kit is designed to quantify the amount of native type II collagen from most species in cell or tissue culture or from tissue homogenates by ELISA.


 Collagen Detection Antibodies and Kits Flyer [PDF]

measurement principle

The Rheumera™ assay measures native type II collagen from cell/tissue culture or cartilage tissue using enzyme-linked immunosorbent assay (ELISA). The kit will measure 80 individual samples or 40 samples in duplicate. Removable strips and aliquoted reagents allow for samples to be tested on partial plates on 2 separate occasions. Samples are incubated in wells that are coated with antibodies to type II collagen. Dilutions of a standard (highly purified type II collagen) is also incubated in wells that are coated with antibodies to type II collagen. The wells are washed to remove unbound type II collagen molecules, and then incubated with antibodies to type II collagen which are conjugated to biotin. The wells are washed to remove unbound antibodies and then incubated with avidin (which has affinity to biotin) conjugated to peroxidase. The wells are washed to remove unbound avidin-peroxidase and then incubated with a chromogen substrate solution, TMB. A blue color develops which then.

[Important] 
Newly synthesized collagen from cells in culture is incorporated into the extracellular matrix and remains in fibril form. Therefore, very little soluble, monomeric collagen exists in culture media or in tissue. Thus, to measure collagen content in cell culture or in tissue using the Native Type II Collagen Detection Kit, the collagen must first be digested with pepsin in acidic conditions and then further digested with pancreatic elastase at neutral pH in order to convert polymeric collagen to monomeric collagen turns yellow when the stop solution, mild sulfuric acid, is added. The color intensity is proportion to the amount of type II collagen present in the sample. The sample values, ng/ml of native type II collagen, are determined by the standard curve.

Standard curve

Kit Contents

A Anti-type II collagen-coated strips 10
B Standard coated strips 4
C Diluent Buffer (Solution) 1X (60ml)
D Wash Buffer (Solution) 10X (50ml)
E Anti-type II collagen (Lyophylized. Conjugated to biotin) 2 vials
F Standard (Purified type II collagen 0.2 ml (1000 ng/ml)) 1 vial
G Sample/Standard Dilution (Solution) 1X (20ml)
H Avidin-Peroxidase (Lyophylized) 2 vials
I TMB (1X). Chromogen substrate for peroxidase detection 1X (10ml)
J Stop Solution (1X).Diluted sulfuric acid to stop the color reaction 1X (5ml)

<Reference>
Eyre, DR, Weis, MA and Wu, JJ. and Articular cartilage collagen: an irreplaceable framework Eur Cell Mater. 2006 Nov 2;12:57-63. Review

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