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

ELISA Assay Kit to measure mouse antibodies to type II collagen

Background

Collagen-induced arthritis (CIA) is a model of chronic inflammatory arthritis which closely resembles human rheumatoid arthritis (RA). Since CIA shares similar immunological and pathological features with RA, this makes it an ideal model for screening therapeutics. This model also has the primary advantage that the mechanisms of pathogenesis are known; susceptible mice, rats or monkeys (Trentham, 1982; Cathcart et al., 1986; Yoo et al., 1988) develop arthritis after immunization with heterologous, native type II collagen. For mice, collagen is emulsified with complete Freund’s adjuvant which is injected s.c. at the base of the tail. The onset of arthritis occurs within 4 to 5 weeks. For rats, collagen is emulsified in incomplete Freund’s adjuvant (since rats are susceptible to adjuvant arthritis) and the onset of arthritis occurs within 10 days to 3 weeks, depending upon the strain of rat used. Clinical signs of arthritis include red and swollen paws. Histopathological features include infiltration of inflammatory cells, pannus formation and cartilage and bone destruction.

The immunopathogensis of CIA is initiated when crossreactive antibodies, mounted against the heterologous type II collagen, bind to the host’s cartilage, activate complement and subsequently the inflammation cascade consisting of pro-inflammatory cytokines, chemokines and matrix matrix-degrading enzymes. Since anti-collagen antibodies are the effectors of inflammation, their presence in serum directly correlates with the onset of arthritis and provides a useful clinical marker. The Rheumera ELISA system allows the measurement of anti-type II collagen antibodies in serum from mice. The kit can be ordered with various species of type II collagen.


 Collagen Detection Antibodies and Kits Flyer [PDF]

measurement principle

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 anti-mouse IgG 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.

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

Standard curve

1. Average the duplicate OD values for the blanks (B), standards and test samples. 

2. Subtract the blank (B) values from the averaged OD values in step 1. The blank (B) wells for each different species of collagen should be used, if more than one species is used.

3. Plot the OD values of standards against the Units/ml of antibody standard. Using a log/log plot will make the data linear. The figure to the right shows a representative experiment where the standard range is from 0 to 100 Units/ml.

4. The Units/ml of antibody in test samples can be calculated using regression analysis

Kit Contents

A Anti-type II collagen-coated strips 10
B Standard coated strips 4
C Blocking/Diluent Buffer (1X) 60ml
D Wash Buffer (10X) 50ml
E Secondary Ab 2 vials
F Standard (Lyophilized) 1 vials
G TMB (1X) (Chromogen substrate) 10ml
H Stop Solution (1X) (Diluted sulfuric acid ) 5ml

<Reference>
Brahn, E. (1991). Animal models of rheumatoid arthritis. Clues to etiology and treatment. Clin Orthop 265, 42-53.

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