Principle of the Assay

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, Rheumera™ 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 Rheumera™ 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.

Reagents Included

1. Type II collagen-coated strips (10). Includes 2 Standard strips (see above chart for color coding).
2. Un-coated strips (10). Includes 2 Standard strips.
3. Blocking Buffer 1X (20ml).
4. Diluent Buffer 1X (60ml)
5. Wash Buffer - 1 tablet. Dissolve tablet in 1000 ml dH₂O
6. Standard (2 vials). Lyophilized. Dilute one vial with 1.0 ml 1X Diluent Buffer (D)
7. Secondary Ab (2 vials). Lyophylized. Anti-human IgG, conjugated to biotin. Dilute one vial in 10.0 ml 1X Diluent Buffer (D).
8. Avidin-peroxidase (2 vials). Lyophylized. Steptavidin-Avidin. Dilute one vial in 10.0 ml 1X Diluent Buffer (D).
9. TMB (1X) - 20ml. Chromogen substrate for peroxidase detection.
10. Stop Solution (1X) – 10 ml. Dilute sulfuric acid to stop the color reaction.

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For Research Use Only, Not for Diagnostic or Therapeutic Use

The kit can be ordered with various species of type II collagen (see chart below).

 

Examples of Different ELISA Plate Configurations

A Typical ELISA plate

Uncoated HII Stnd A typical ELISA plate where samples and standards are run in duplicate. Five, 8-well strips are uncoated, depicted in white circles, and serve as controls for sample background levels.

Five additional 8-well strips are coated with one species of type II collagen (such as human; HII depicted in blue) to determine specific antibody levels. Two, 8-well strips are used for the standards (0 to 100 units/ml), depicted in red. “B” represents blank wells to determine secondary antibody background levels.

 

 

Custom ELISA plate

Uncoat. HII CII BII PII Stnd A custom ELISA plate where samples and standards are run in duplicate. Two, 8-well strips are uncoated and serve as controls for sample background levels.

Two each, 8-well strips are coated with multiple species of type II collagen, such as human (HII), in blue, chick (CII) in yellow, bovine (BII) in green and porcine (PII) in purple to determine specific antibody levels. Two, 8-well strips are used for the standards (0 to 100 units/ml) depicted in red. “B” represents blank wells to determine secondary antibody background levels

 

 

Select the species of type II collagen  (custom combinations are availalbe upon request)

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