Intended Use
Rheumatoid Factor IgA/IgG/IgM ELISA is a solid phase enzyme immunoassay with highly purified Fc fragment of human immunoglobuline (IgG) for the separate quantitative and qualitative detection of IgG, IgM and IgA rheumatoid factors (RF) in human serum.
Contents of Kit
1. Sample Buffer (5×): 1 × 20 mL. 5 × concentrated. Tris, sodium chloride (NaCl), bovine serum albumin (BSA), sodium azide < 0.1% (preservative).
2. Wash Buffer (50×): 1 × 20 mL. 50 × concentrated. Tris, NaCl, Tween 20, sodium azide < 0.1%(preservative)
3. Negative Control: 1 × 1.5 mL. Human serum (diluted), bovine serum albumin (BSA), sodium azide < 0.1% (preservative)
4. Positive Control: 1 × 1.5 mL. Human serum (diluted), bovine serum albumin (BSA), sodium azide < 0.1% (preservative)
5. Cut-off Calibrator: 1 × 1.5 mL. Human serum (diluted), bovine serum albumin (BSA), sodium azide < 0.1% (preservative)
6. Calibrators: 6 × 1.5 mL. Concentration of each calibrator: 0, 3, 10, 30, 100, 300 U/ml. Human serum (diluted), bovine serum albumin (BSA), sodium azide < 0.1% (preservative).
7. Conjugate, IgG: 1 × 15 mL.
Conjugate, IgM: 1 × 15 mL.
Conjugate, IgA: 1 × 15 mL.
Containing: Anti-human immunoglobulins conjugated to horseradish peroxidase, bovine serum albumin (BSA)
8. TMB Substrate: 1 × 15 mL.
9. Stop Solution: 1 × 15 mL. 1M Hydrochloric Acid
10. Microtiter plate: 12 × 8 well strips. With breakaway microwells. Refer to paragraph 1 for coating.
Storage
Store all reagents and the microplate at 2-8°C/35-46°F, in their original containers. Once prepared, reconstituted solutions are stable at 2-8°C/35-46°F for at least 1 month. Reagents and the microplate shall be used within the expiry date indicated on each component, only. Avoid intense exposure of TMB solution to light. Store microplates in designated foil, including the desiccant, and seal tightly.
Performance Characteristics
Sample material: serum
Sample volume: 10 μl of sample diluted 1:101 with 1x sample buffer
Total incubation time: 90 minutes at 20-32°C/68-89.6°F
Calibration range: 0-300 U/ml
Analytical sensitivity: 1.0 U/ml
Storage: at 2-8°C/35-46°F use original vials only.
Calibration: Due to the lack of international reference calibration this assay is calibrated in arbitrary units (U/ml) for IgG and IgA rheumatoid factors. For IgM rheumatoid factor, the assay is calibrated against the international WHO standard and results are given in IU/ml.
Precision
To determine the precision of the assay, the variability (intra and inter-assay) was assessed by examining its reproducibility on three serum samples selected to represent a range over the standard curve.
Sensitivity
Analytical sensitivity: Testing sample buffer 30 times on Rheumatoid Factor IgA/IgG/IgM ELISA gave an analytical sensivity of 1.0 U/ml.
Specificity and sensitivity: The microplate is coated with Fc fragment of human immunoglobuline (IgG). No crossreactivities to other autoantigens have been found. Rheumatoid factors are detected in 70-90% of patients with rheumatoid arthritis (RA).
General Description
Rheumatoid factors (RF), first described in 1940 as antibodies reacting with gamma globulins, are autoantibodies directed against the C-terminal part of the constant region of the IgG heavy chain, the IgG Fc.
Although named after the disease they were initially associated with, RFs are found both in the healthy population and several diseases. The diseases commonly associated with high RF concentrations are rheumatoid arthritis (RA; 50-90%) and Sjögren`s syndrome (75-95%).
They are also found in systemic lupus erythematosus (SLE; 15-35%), systemic sclerosis (20-30%) polymyositis / dermatomyosisits (5-10%), cryoglobulinemia (40-100%) and mixed connective tissue diseases (MCTD; 50-60%).
Although the presence of IgM RF in the serum has been regarded as the most important serological indicator for RA, thus included in the ACR list of criteria for the diagnosis of this disease, RF of IgG and IgA subclass are important for diagnosis as well.
Determination of these isotypes provides additional information with regard to diagnosis, differential diagnosis and follow-up of RA in comparison to conventional techniques such as latex agglutination test and nephelometry. Whilst RF of subclass IgM are most sensitive for diagnosis of RA, thus most suitable for screening, RF of subclass IgG are most specific for RA and like subclass IgA correlate with clinical parameters and disease activity. The presence of all three subclasses together is 100 % specific for RA.
RF in SLE are associated with sicca syndrome, hypergammaglobulinemia, high titer of antinuclear antibodies, anemia and usually SS-A and SS-B antibodies appearance. All three subclasses are found in SLE. Especially the subclass IgA defines a subgroup of SLE patients characterized by distinct autoimmune phenomena and high disease activity in the absence of nephritis.
Standard Curve
Example of a standard curve Do NOT use this example for interpreting patient's result
| Calibrators IgG/A/M | OD 450/620 nm | CV % (Variation) |
| 0 U/ml | 0.035 | 2.3 |
| 3 U/ml | 0.138 | 2.6 |
| 10 U/ml | 0.342 | 3.2 |
| 30 U/ml | 0.632 | 3.2 |
| 100 U/ml | 1.216 | 0.5 |
| 300 U/ml | 2.178 | 0.1 |
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