Anti-Cyclic Citrullinated Peptides (CCP) IgG ELISA Kit

Sample

Serum

Species Reactivity

Human

Detection Method

iELISA

Intended Use

Anti-Cyclic Citrullinated Peptides (CCP) IgG ELISA Kit is an enzyme-linked immunosorbent assay (ELISA) for qualitative and semi-quantitative determination of IgG antibodies to Cyclic Citrullinated Peptides (CCP) in human sera. The assay is used to detect antibodies in a single serum specimen. For research use only.

Contents of Kit

1. CCP peptide-coated microtitre plate: 96 wells (Sealed). Ready to use.
2. Calibrators (positive human serum pool): 1.2 mL × 5 Vials. Ready to use (blue).
3. Reference control human serum: 1.2 mL × 1 Vial. Ready to use (blue).
4. Positive control human serum: 1.2 mL × 1 Vial. Ready to use (blue).
5. Negative control human serum: 1.2 mL × 1 Vial. Ready to use (blue).
6. Conjugate solution (peroxidase conjugated to anti human IgG antibodies: 15 mL × 1 Vial. Ready to use (red).
7. Substrate solution TMB: 15 mL × 1 Vial. Ready to use.
8. Dilution buffer: 35 mL × 2 Vials. Ready to use (blue).
9. Stop solutio: 15 mL × 1 Vial. Ready to use.
10. Wash buffer: 35 mL × 2 Vials. 20 × concentrated.

Storage

1. Store the kit at + 2° C to + 8° C in a dark place.
2. Do not use reagents beyond their expiration date.
3. It is advisable to unpack the sealed microtitre plate immediately before use.
4. Any direct action of light on the chromogen solution should be avoided.
Note: If a weak or absent colour reaction of the first calibrator A (3200 U/mL) E450 nm < 0.9, is observed, the test result is invalid.

Performance Characteristics

The anti-CCP EIA measures antibodies against synthetic peptides with citrulline residues. The anti CCP EIA is calibrated in the semi-quantitative assay in relative units using a positive patient serum pool. The standard curve ranges 25-3200 U/mL. These values have been chosen arbitrarily by Creative Diagnostics since no generally recognised international standard exists for expressing the titre of anti-CCP antibodies. The specificity and sensitivity were evaluated in previous studies with 311 RA patients, 942 diseased non-RA patients (including other autoimmune and wide range of infectious diseases) and 330 healthy controls. The sensitivity was 70%. The specificity was 97% with diseased non-RA patients and 99% with healthy individuals.

Precision

Intra-assay precision was determined by testing six different samples eight times each.

Inter-assay precision was determined by testing six different samples eight times each. Results were obtained for three different runs.

Lot to lot variation was determined by testing six different samples eight times each. Results were obtained for three different lots.

Detection Limit

The detection limit of the assay was determined by running the zero standard 14 times on three different lots. The detection limit of 1.6 U/mL was calculated by finding the mean plus two standard deviations.

General Description

Rheumatoid Arthritis (RA) is one of the most common systemic autoimmune diseases. The aetiology of the disease, which affects up to 1-2% of the world population, is unknown. The diagnosis of RA depends primarily on clinical manifestation of the disease. The only serological test routinely used is the determination of the presence of rheumatoid factors (RF) in the serum. RF are antibodies directed to the constant region of immunoglobulins of the IgG class. However, these antibodies are also present in relatively high percentages in other autoimmune diseases, infections and in up to 15% of healthy individuals.
Antibodies of a more specific nature have also been found in sera of RA patients. Anti-perinuclear factor (APF) antibodies are reported to be present in around 50% of RA patients with a specificity of over 70%. A number of cyclic synthetic peptides not related to filaggrin or other known proteins were described which are specifically recognized by autoantibodies in sera from RA patients. These peptides were subsequently used in an EIA for the detection of RA-specific autoantibodies. Clinical evaluation studies showed that the EIA was positive in a significant number of well-defined RA patient sera with an excellent specificity against disease controls. A diagnostic and prognostic value for the measurement of the anti Cyclic Citrullinated Peptides (anti-CCP) antibodies was found in relation to joint involvement and radiological damage in early RA. Anti-CCP antibodies can be detected years before the development of clinical symptoms. A prospective cohort study showed that 93% of the anti-CCP positive patients with undifferentiated arthritis finally developed rheumatoid arthritis, demonstrating the strong positive predictive value of these antibodies. The Anti-Cyclic Citrullinated Peptides (CCP) IgG ELISA Kit offered by Creative Diagnostics is based on highly purified synthetic peptides containing citrulline residues and is a valuable addition to the diagnosis of RA. This anti-CCP kit contains improved synthetic peptides selected on the basis of superior performance in the detection of RA autoantibodies.