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The Rheumera™ ELISAsystem allows the measurement of anti-type II collagen antibodies in serum from human.

ELISA Assay Kit to measure human antibodies to type II collagen


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Background

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints, which ultimately results in the destruction of cartilage and bone. Components of the inflammatory process include cells, cytokines and matrix metalloproteinases. Although the etiology of the disease is unknown, one potential autoantigen, type II collagen, has been implicated in RA. Type II collagen is the major protein of articular cartilage and consists of three pro-alpha1(II) chains twisted together to form a triple-stranded helical molecule.

Autoantibodies to type II collagen have been widely reported in adult and juvenile rheumatoid arthritis and relapsing polychondritis (see references). In these patients, antibodies have been found in serum, synovial fluid and eluted from cartilage explants. It is hypothesized that antibodies to type II collagen are involved in the pathogenesis of RA because 1) Type II collagen is highly immunogenic and immunization of susceptible mice, rats and non-human primates results in inflammatory arthritis, known as collagen-induced arthritis (CIA). 2) Antibodies to type II collagen are found in the serum of CIA animals and can be passively transferred to naïve recipients to induce disease and 3) antibodies to type II collagen found in RA are complement-fixing. Antibodies to type II collagen have also been found in other diseases such as relapsing polychondritis (RP), SLE, scleroderma, diseases of the heart and vasculature and otosclerosis.


 Collagen Detection Antibodies and Kits Flyer [PDF]

Measurement principle

Research has determined that antibodies to type II collagen from patients with RA or RP react to various species of type II collagen such as chicken, bovine and porcine (see references). It has been hypothesized that antibodies are generated to these various species of dietary collagen which crossreact to human collagen, possibly inducing disease if they bind to cartilage, activate complement and subsequently the inflammation cascade. Added proof for this hypothesis comes from studies showing antibody reactivity to various species of type II collagen in healthy persons. Therefore, RheumeraTM ELISA kits can be ordered with various species of type II collagen (see table below).

One of the challenges of measuring antibodies from serum of patients with autoimmune disease, is that it contains high levels of protein components which bind to ELISA wells whether they are coated or uncoated. This results in false positive reactions yielding high background levels. The RheumeraTM ELISA kits use 2 methods to deal with false positive reactions. First, a unique blocking buffer is applied to the ELISA wells which inhibit non-specific binding. Secondly, samples are incubated in uncoated wells to determine background levels. These background levels are then subtracted from their collagen-coated counterparts, to determine specific antibody binding.

This assay measures antibodies to native human type II collagen from serum or synovial fluid using enzyme-linked immunosorbent assay (ELISA). The kit will measure up to 39 samples in duplicate. Removable strips and aliquoted reagents allow for samples to be tested on 2 partial plates on 2 separate occasions.

Samples are incubated in wells that are uncoated or coated with type II collagen which have been first pre-treated with blocking buffer. Standards are incubated in wells that are coated with type II collagen. The wells are washed to remove unbound antibodies, and then incubated with an anti-human IgG antibody, conjugated with a biotin label. The wells are washed to remove unbound antibodies and then incubated with avidin (which binds to biotin), conjugated to peroxidase. The wells are washed to remove unbound avidin-peroxidase, then incubated with a chromogen substrate solution, TMB. A blue color develops which then turns yellow when the stop solution, mild sulfuric acid, is added. The color intensity is proportion to the amount of anti-collagen antibody bound to type II collagen The sample values, measured as Units/ml, are determined by the standard curve.

Standard curve

Kit Contents

A Anti-type II collagen-coated strips (Includes 2 Standard strips) 10
B Un-coated strips ( Includes 2 Standard strips) 10
C Blocking Buffer (1X) 20ml
D Diluent Buffer (1X) 60ml
E Wash Buffer (dissolve tablet in 1000 ml dH2O) 1 tablet
F Standard (Lyophilized) 2 vials
G Secondary Atybody (Lyophilized) 2 vials
H Avidin-Peroxidase (Lyophylized) 2 vials
I TMB (1X) (Chromogen substrate) 20ml
J Stop Solution (1X) (Diluted sulfuric acid ) 10ml

<Reference>
R. B. Clague, K. Morgan, T. I. Reynolds, H. J. Williams. The prevalence of serum IgG antibodies to Type II Collagen in American patients with rheumatoid arthritis. British Journal of Rheumatology, 1994;33:336-338

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References

R. B. Clague, K. Morgan, T. I. Reynolds, H. J. Williams. The prevalence of serum IgG antibodies to Type II Collagen in American patients with rheumatoid arthritis. British Journal of Rheumatology, 1994;33:336-338

MJ Rowley, B Tait, T Doran, P Emery and IR Mackay. Associations between HLA and antibodies to collagen in rheumatoid arthritis. Annals of the Rheumatic Diseases, 1990, Vol 49, 578-581.

Kim W.-U., Yoo W.-H., Won Park, Kang Y.-M., Kim S.-I., Park J.-H., Lee S.-S., Joo Y.-S, Min J.-K., Hong Y.- S., Lee S.-H., Park S.-H., ChoC.-S., Kim H.-Y. IgG antibodies to type II collagen reflect inflammatory activity in patients with rheumatoid arthritis. Journal of Rheumatology, 2000; 27(3):575-581.

Rowley MJ, Williamson DJ, Mackay IR Evidence for local synthesis of antibodies to denatured collagen in the synovium in rheumatoid arthritis. Arthritis Rheum. 1987; 30(12):1420-5

Terato K., DeArmey D.A., Ye X.J., Griffiths M.M., Cremer M.A. The Mechanism of Autoantibody Formation to Cartilage in Rheumatoid Arthritis: Possible Cross-Reaction of Antibodies to Dietary Collagens with Autologous Type II Collagen. Clin Imm Immunopath, 1996; 79(2):142-154(13)
Terato K, Shimozuru Y, Katayama K, et al. Specificity of antibodies to type II collagen in rheumatoid arthritis. Arthritis Rheum 1990;33:1493-500.

Andriopoulos NA, Mestecky J, Miller EJ, Bennett JC. Antibodies to human native denatured collagen in synovial fluid of patients with rheumatoid arthritis. Clin Immunol Immunopathol 1976;6:209-12.

Clague RB, Moore LJ. IgG and IgM antibody to native type II collagen in rheumatoid serum and synovial fluid: evidence for the presence of collagen-anticollagen immune complexes in synovial fluid. Arthritis Rheum 1984;27:1370-7.

Fujii K, Tsuji M, Kitamura A, Murota K. The diagnostic significance of anti-type II collagen antibody assay in rheumatoid arthritis. Int Orthop 1992;16:272-6.

Cook AD, Rowley MJ, Stockman A, Muirden KD, Mackay IR. Specificity of antibodies to type II collagen in early rheumatoid arthritis. J Rheumatol 1994;21:1186-91.

Mullazehi M; Mathsson L; Lampa J; Ronnelid J: Surface-bound anti-type II collagen-containing immune complexes induce production of tumor necrosis factor alpha, interleukin-1beta, and interleukin-8 from peripheral blood monocytes via Fc gamma receptor IIA: a potential pathophysiologic mechanism for humoral anti-type II collagen immunity in arthritis. Arthritis Rheum. 2006 Jun;54(6):1759-71

Mullazehi M, Mathsson L, Lampa J, Ronnelid J: Anti-collagen type-II antibody levels and induction of proinflammatory cytokines by anti-collagen antibody-containing immune complexes in vitro characterise a distinct rheumatoid arthritis phenotype associated with acute inflammation at the time of disease onset. Ann Rheum Dis. 2007 Apr;66(4):537-41

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