REAADS Anti-Cardiolipin (aCL) IgA

REAADS Anti-Cardiolipin (aCL) IgA is an ELISA for the determination of immunoglobulin A anti-cardiolipin antibodies in human serum of plasma in subjects with system lupus erythematosus (SLE), lupus-like disorders (anti-phospholipid syndrome) and other auto-immune diseases. Anti-cardiolipin antibodies are associated with arterial and venous thrombosis, thrombocytopenia and recurrent fetal loss. Presented in automation friendly format (30’ – 30’ – 30’).

Reagents

Each REAADS IgA Anti-Cardiolipin 96-microwell Test Kit contains the following reagents:

(volumes may vary depending on kit size and configuration)

• 96 stabilized beef heart cardiolipin (diphosphatidyl glycerol) coated microwells (12 strips of 8 breakaway wells), with frame.
• 1 bottle (60mL) Sample Diluent* (green solution); contains bovine calf serum.
• 3 vials (0.250mL) aCL IgA Calibrator Serum* (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.
• 1 vial (0.250mL) aCL IgA Positive Control Serum* (human); see vial label for expected APL range.
• 1 vial (0.250mL) aCL Normal Control Sera* (human); see vial label for expected APL range.
• 1 bottle (15mL) anti-human IgA (goat) HRP-conjugated antibody solution (orange solution).
• 1 bottle (15mL) One Component Substrate (TMB and H2O2); ready to use.
• 1 bottle (15mL) Stopping Solution (0.36 N sulfuric acid).
• 2 bottles (30mL) Wash Concentrate (33X PBS).

Test Principle

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.

Protocol Summary

1. Add 100µl of calibrator, controls and samples to microwells
2. Incubate at room temperature for 15 minutes
3. Decant contents
4. Wash 5 times with PBS
5. Add 100µl of conjugate to each well except the water blank
6. Incubate at room temperature for 15 minutes
7. Decant contents
8. Wash 5 times
9. Add 100µl of substrate to each well except the water blank
10. Incubate at room temperature for 10 minutes
11. Add 100µl of stop solution to each well except the water blank
12. Add 200µl of distilled or deionized water to the water blank well
13. Read the O.D of each well at 450nm against a 650nm reference filter (if available) within 30 minutes

Background

Anti-phospholipid antibodies are autoantibodies that react with most negatively charged phospholipids, including cardiolipin (CL). Additionally, anti-phospholipid antibodies are known to prolong in vitro phospholipid-dependent coagulation tests and have been historically referred to as the “lupus anticoagulant”. Paradoxically, patients with the lupus anticoagulant do not present with abnormal bleeding except in the presence of other hemostatic abnormalities.

Anti-cardiolipin (aCL) antibodies are frequently found in patients with systemic lupus erythematosus (SLE). They are also found in patients with other autoimmune diseases, as well as in some individuals with no apparent previous underlying disease. Elevated levels of aCL antibodies have been reported to be significantly associated with the presence of both venous and arterial thrombosis, thrombocytopenia, and recurrent fetal loss. The term “anti-phospholipid syndrome” (APS) has been introduced to describe patients who present these clinical manifestations, in association with aCL antibodies or the lupus anticoagulant.

High serum levels of IgG aCL antibodies are more prevalent and more clinically relevant than IgM aCL antibodies when correlated with clinical manifestations of the APS. Studies indicate that elevated serum levels of IgA aCL antibodies are also frequently found in patients with SLE and related disorders. IgA aCL serum levels were significantly higher in SLE patients with vascular complications than those without, and correlated with a predisposition to thrombosis, thrombocytopenia, and fetal loss.

The REAADS IgA Anti-Cardiolipin Test Kit uses a well-known ELISA format capable of detecting a specific isotype of aCL antibodies in human serum or plasma. Solid-phase immunoassays are generally considered more sensitive and more specific for detecting aCL antibodies than coagulation assays. The REAADS IgA Anti-Cardiolipin Test Kit provides rapid, highly reproducible, accurate, and objective results in units that have been standardized against a reference preparation. The values for IgA aCL antibodies are reported in APL (IgA antiphospholipid) units.