REAADS Anti-Cardiolipin (aCL) IgA is an ELISA for the determination of IgA anti-cardiolipin antibodies in human serum or plasma in individuals with systemic lupus erythematosus (SLE) and lupus-like disorders (anti-phospholipid syndrome).
- Specific determination of IgA aCL antibodies
- Convenient ELISA procedure
- Objective, accurate and reproducible
- Reagent complete kit
- Short incubations at room temperature
Reagents, Storage and Stability
- Stabilized beef heart cardiolipin (diphosphatidyl glycerol) coated microwells: 96 (12 strips of 8 breakaway wells), with frame.
- Sample Diluent: 1 bottle, 60 ml (green solution), contains bovine calf serum and sodium azide.
- aCL IgA Calibrator Serum: 3 vials, 0.250 ml (1-high, 2-moderate, 3-low) (human), see vial label for antibody concentration in APL units. Calibrator 2 should be used when performing single point calibration. Contains sodium azide.
- aCL IgA Positive Control Serum: 1 vial, 0.250 ml (human), see vial label for expected APL range. Contains sodium azide.
- aCL Normal Control Sera: 1 vial , 0.250 ml, (human), see vial label for expected APL range. Contains sodium azide.
- anti-human IgA (goat) HRP-conjugated antibody solution: 1 bottle, 15 ml (orange solution).
- One Component Substrate: 1 bottle, 15 ml (TMB and H2O2), ready to use.
- Stopping Solution: 1 bottle, 15 ml (0.36 N sulfuric acid).
- Wash Concentrate 2 bottles, 30 ml (33X PBS).
Store at 2 – 8°C. Do Not Freeze.
The test is performed as an indirect ELISA. Diluted serum samples, calibrator sera, and controls are incubated in cardiolipin coated microwells, allowing aCL antibodies present in the samples to react with the immobilized antigen. After the removal of unbound serum proteins by washing, antibodies specific for human IgA labeled with horseradish peroxidase (HRP) are added forming complexes with the cardiolipin bound antibodies. Following another washing step, the bound enzyme-antibody conjugate is assayed by the addition of tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) as the chromogenic substrate. Color develops in the wells at an intensity proportional to the serum concentration of IgA aCL antibodies. Results are obtained by reading the O.D. (optical density or absorbance) of each well with a spectrophotometer. Calibrator sera are provided, with the IgA aCL concentration expressed in APL units. The user has the option of running either a single point calibrator or a four-point calibration curve. For single point calibration, dividing the concentration value of the calibrator by the O.D. value of the calibrator provides a conversion factor. The O.D. values of the controls and patient samples are multiplied by the conversion factor to obtain IgA aCL values, expressed in APL units. For multipoint calibration, perform a linear regression analysis with calibrator values against calibrator O.D.s. Control and patient results are determined from the calibration curve.
Diluted patient serum (preferred) or plasma is incubated in coated microwells. Cardiolipin antibodies present in the sample will bind to the coated wells. After washing to remove unbound plasma proteins, enzyme conjugated anti-human immunoglobulin specific for IgA is added. The wells are washed again, and a chromogenic substrate is added, resulting in a colored product. The reaction is stopped by the addition of a weak acid solution, and the colored product is measured in a spectro-photometer at 450 nm. Test results are available in less than one hour. Values for aCL antibodies are reported in GPL, MPL, and APL units, with assay cutoffs established at 23 GPL, 11 MPL, and 22 APL.
Clinical Specificity: Assay cutoffs were challenged with a healthy blood donor population. Using the stated cutoffs, the assays were 97% specific for IgG, 96% specific for IgM, and 95% specific for IgA.
Unselected SLE populations were tested to determine assay sensitivity; 21% of the samples were positive for IgG, 10% for IgM and 26% for IgA antibodies with the REAADS aCL assays. The clinical sensitivity for thrombosis and thrombocytopenia was determined by comparing aCL test results from two groups of selected SLE patients: Group 1 ? with a clinical history of thrombosis and/or thrombocytopenia; and Group 2 ? with no history of thrombosis or thrombocytopenia (control). The results are shown in the charts below:
|Group 1: SLE + Thrombosis and/or thrombocytopenia||IgG aCL||IgM aCL||IgA aCL|
|Average value||30 GPL||9 MPL||32 APL|
|Group 2: SLE Control||IgG aCL||IgM aCL||IgA aCL|
|Average value||8 GPL||3 MPL||15 APL|
|K023-001||IgG / IgM||96||rapid format (15-15-10)|
|K023-002*||IgG / IgM||288||rapid format (15-15-10)|
|K026-001||IgA||96||rapid format (15-15-10)|
|K026-006*||IgA||288||rapid format (15-15-10)|
|K11139*||IgG||96||extended incubations (30-30-30)|
|K12747*||IgG||288||extended incubations (30-30-30)|
|K11140*||IgM||96||extended incubations (30-30-30)|
|K12748*||IgM||288||extended incubations (30-30-30)|
|K11141*||IgA||96||extended incubations (30-30-30)|
|K12770*||IgA||288||extended incubations (30-30-30)|
* This product may require a special order. Please inquire at firstname.lastname@example.org or 1-800-526-5224.
Anti-cardiolipin (aCL) antibodies are associated with the presence of both venous and arterial thrombosis, thrombocytopenia, and recurrent fetal loss. These autoantibodies are frequently found in patients with systemic lupus erythematosus (SLE) and other autoimmune diseases, as well as in some individuals with no apparent previous underlying disease.
|What is the clinical significance when samples test positive for the anti-cardiolipin antibodies and are negative for anti-b2GPI?|
|The aCL assay can detect antibodies of differing specificities. This includes antibodies specific for the cardiolipin molecule itself, and antibodies that are directed against either a cofactor molecule such as b2GPI, or a special binding site created by the interaction of a cofactor with CL. Antibodies directed against CL may be associated with infectious disease, or may be specific for a different cofactor, such as prothrombin. The clinical significance of these antibodies must be assessed in conjunction with the patient’s symptoms, clinical history, and other laboratory findings. Follow-up testing of these patients is recommended in 3-6 months to confirm reactivity. Only b2GPI cofactor dependent antibodies react in the anti-b2GPI assay; these antibodies show a higher correlation with thrombosis and are more specific for the antiphospholipid syndrome.|
|What are some features of the REAADS anti-Cardiolipin test? The b2GPI test?|
|The REAADS anti-Cardiolipin test kit and the b2GPI kit are reagent-complete kits. The anti-cardiolipin kit features specific determination of IgG, IgM, and IgA aCL antibodies. The kits are convenient, cost-effective ELISA procedures which give objective, accurate, and reproducible results with short incubations at room temperature.|
|How does a syphilis infection affect the REAADS anti-cardiolipin test?|
|Patients with current or prior syphilis infections may have a positive result without increased risk of thrombosis. Anti-cardiolipin antibodies can appear transiently at low levels during many infections. If a patient first tests positive while there are clinical signs of infection, the test should be repeated after an interval of six months.|
|Summarize the IgG/IgM and IgA REAADS anti-cardiolipin test. What is the normal range?|
|The test is an indirect ELISA. Diluted serum samples, calibrator sera, and controls are incubated in cardiolipin coated microwells, allowing aCL antibodies present in the samples to react with the immobilized antigen. After their removal of unbound serum proteins by washing, antibodies specific for human IgG, IgM, or IgA labeled with HRP are added forming complexes with the CL bound antibodies. Following another washing step, the bound enzyme-antibody conjugate is assayed by the addition of TMB and H2O2 as the chromogenic substrate. Color develops in the wells at an intensity proportional to the serum concentration of aCL antibodies. The OD is read at 450 nm.
Normal ranges are less than 23 GPL (IgG per liter), less than 11 MPL (IgM per liter), and 22 APL (IgA per liter).