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Technical Information

1.Rheumatoid Factor antibodies
The Rheumatoid Factor ELISA kits are rapid methods for the detection of total or class-specific rheumatoid factor. They are intended as an aid to the diagnosis of rheumatoid diseases. The components of the kits are for in vitro diagnostic use only.

2. Explanation of the Test
Rheumatoid factors (RFs) are autoantibodies directed against antigenic sites in the Fc region of human IgG.  Elevated RFs are found in 70-90% of patients with rheumatoid arthritis (RA) and also occur frequently in patients with other rheumatic as well as infectious and pulmonary diseases.

Traditional agglutination methods measure principally IgM antibodies. However, patients may have IgA, IgG or IgM RFs, either alone or in combination.  Elevated concentrations of IgM RFs are found in approximately 70-80% of patients with confirmed RA and correlate with disease activity and vasculitis.   IgM RFs are considered a risk factor in normal subjects.  IgA RFs are reportedly associated with bone erosions and symptoms originating from mucosal membranes and secretory organs.  A number of studies indicate that the IgA RFs in early disease indicate poor prognosis and justify a more aggressive course of treatment.  Elevation in IgA RFs may precede the increase in IgM RF titre. Elevated concentrations of IgG RFs may precede the development of RA and are also considered a risk factor in normal subjects.  Raised IgG RFs are virtually confined to the sera of patients with RA and not other arthritides.

3. Principle of the test
Diluted serum samples are incubated with purified rabbit immunoglobulin IgG immobilised on microtitre wells.  After washing away unbound serum components, anti-human IgA-G-M (GD06), IgM (GD23), IgA (GD24) or IgG (GD37) peroxidase conjugate is added to the wells and this binds to surface-bound antibodies in the second incubation. Unbound conjugate is removed by washing, and a solution containing 3,3’,5,5’-tetramethylbenzidine (TMB) and enzyme substrate is added to trace specific antibody binding. Addition of Stop Solution terminates the reaction and provides the appropriate pH for colour development.  The optical densities of the standards, controls and samples are measured using a microplate reader at 450nm. Optical density is directly proportional to antibody activity in the sample. 

Citrullinated protein Antigen (CPA)

1.Citrullinated protein antigen
The Citrullinated Protein Antibodies kit is a rapid ELISA method for the detection of rheumatoid arthritis specific IgG antibodies to citrullinated protein.   The components of the kit are for in vitro diagnostic use only.

2. Explanation of the Test
Rheumatoid arthritis (RA) is diagnosed primarily on clinical disease manifestations, and serological supporting evidence has, until recently, been restricted to the determination of rheumatoid factor. However, this antibody occurs in many inflammatory diseases and in healthy, elderly individuals. 

Recently, citrullinated protein antibodies (CPA) have been shown to be highly specific for RA. (1, 2).  The antigenic determinants recognised by these antibodies contain a modified form of arginine, termed citrulline, involved in a peptidic link (1,2). The enzyme responsible for the citrullination is peptidylarginine deiminase (3). 

Citrullination has been shown to be an essential step in generating RA-specific antibodies in susceptible individuals, perhaps via a breakdown in immune tolerance to self-antigens.  Importantly, citrullinated fibrin has been identified in rheumatoid synovial membranes (4). Citrullinated protein is also present in perinuclear granules of buccal mucosa and is associated with keratin fibrils in the stratum corneum of the oesophagus.  Recent studies have shown the citrullinated protein to be filaggrin and the target recognised by anti-perinuclear factor antibodies and the anti-keratin antibodies. 

Importantly, CPA can be detected both in early and fully developed RA and are even present in some patients several years prior to the onset of disease.  The ability to accurately diagnose RA in the early stages of the disease, or even before disease onset, obviously allows for earlier treatment and the opportunity to delay disease progression.

The CPA ELISA uses citrullinated recombinant rat filaggrin as the antigen for the detection of anti-citrullinated protein antibodies because it has been shown to be a more effective substrate than human proteins.   The resulting CPA test is highly specific and sensitive for the detection of RA.

3. Principle of the test
Diluted serum samples are incubated with recombinant citrullinated rat filaggrin immobilised on microtitre wells.  After washing away unbound serum components, rabbit anti-human IgG conjugated to horseradish peroxidase is added to the wells and this binds to surface-bound antibodies in the second incubation. Unbound conjugate is removed by washing, and a solution containing 3,3’,5,5’-tetramethylbenzidine (TMB) and enzyme substrate is added to trace specific antibody binding. Addition of Stop Solution terminates the reaction and provides the appropriate pH for colour development.  The optical densities of the standard(s), controls and samples are measured using a microplate reader at 450nm.  Optical density is directly proportional to the concentration of citrullinated protein antibodies in the sample. 

References

Rheumatoid Factor
Heimer R, Levin FM (1966) On the distribution of rheumatoid factor among the immunoglobulins. Immunochemistry, 3, 1-10

Dunne JV, Carson DA, Speileberg HL, Alspaugh MA & N Vaughan JH (1979) IgA rheumatoid factor in serum and saliva of patients with rheumatoid arthritis and Sjogren’s syndrome. Ann Rheum Dis , 38, 161-166

Gioud-Paquet M,Auvinet M, Raffin T, Hazleman BL (1985) IgM rheumatoid factor (RF), IgA RF IgE RF and IgG RF detected by ELISA in rheumatoid arthritis. Ann Rheum Dis 46, 65 – 71

Annals of the Rheumatic Diseases, 1985 44: 14-19

Annals of the Rheumatic Diseases, 1984, 43: 673-678 & 679-685
.
Annals of the Rheumatic Diseases, 1987 46, 380-384.

Scand J Rheumatology 1988; Supp. 75: 238-243

Scand J Rheumatology 1992; 21: 1-4.

Annals of the Rheumatic Diseases, 1992, 51: 863-868.

The Journal of Rheumatology 1995 22: 571-572

CPA
Schellekens, GA et al:  Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies.  J Clin Invest 1998, 101:23-281

Schellekens, GA et al: The diagnostic properties of rheumatoid arthritis antibodies recognising a cyclic citrullinated peptide. 
Arthritis Rheum 2000, 43: 155-163

Tarcsa, E et al: Protein unfolding by peptidylarginine deiminase.
J Biol Chem 1996, 271: 30709 - 30716

Masson-Bessiere et al: Synovial target antigens of antifilaggrin autoantibodies are deiminated forms of fibrin alpha and beta chains.  Rev Rheum 1999, 66: 754

Vincent C., et al:  Detection of antibodies to deiminated recombinant rat filaggrin by ELISA.  A highly effective test for the diagnosis of rheumatoid arthritis 

Arthritis and Rheumatism 2000, 46;8: 2051