Principle of the Assay

This assay measures antibodies to native type II collagen from serum using enzyme- linked immunosorbent assay (ELISA). The kit will measure 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 coated with type II collagen which have been first pre-treated with blocking buffer. Standards are also incubated in wells that are coated with type II collagen. The wells are washed to remove unbound antibodies, and then incubated with a secondary antibody which is conjugated to peroxidase. The wells are washed to remove unbound secondary antibodies and then incubated with a chromogen substrate solution, TMB. A blue color develops which then turns yellow when the stop solution, dilute 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). See above chart for color coding.
2. Standard coated strips (4).
3. Blocking/Diluent Buffer 1X (60ml). Solution
4. Wash Buffer 10X (50ml). Solution
5. Secondary Ab (2 vials). Lyophylized. Anti-rat IgG, conjugated to peroxidase.
6. Standard (1 vial). Lyophylized.
7. TMB (1X) - 10ml. Chromogen substrate for peroxidase detection.
8. Stop Solution (1X) – 5ml. Dilute sulfuric acid to stop the color reaction.

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For Research Use Only, Not for Diagnostic or Therapeutic Use

Kits can be ordered with single or multiple species of type II collagen as depicted in the following chart:

 

 

Examples of Different ELISA Plate Configurations

Typical ELISA plate

 

1. A standard ELISA plate where samples and standards are run in duplicate. Ten, 8-well strips are coated with the species of type II collagen (in this example, chicken) used for immunization. Two, 8-well strips are used for the standards (0 to 100 Units/ml). “B” represents blank wells to determine secondary antibody background levels.

 

 

 

 

 

A Custom ELISA plate

 

A custom ELISA plate where samples and standards are run in duplicate. Five, 8-well strips are coated with the species of type II collagen (in this example, chicken) used for immunization. Five, 8-well strips are coated with rat type II collagen to measure autoreactive antibodies. Two, 8-well strips are used for the standards (0 to 100 Units/ml). “B” represents blank wells to determine secondary antibody background levels.

 

 

 

For custom orders please contact us.

 

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