DiaMetra Anti Cardiolipin IgM Elisa Kit

CLINICAL SIGNIFICANCE
Cardiolipin is a negatively charged phospholipid which is typically located in the inner mitochondrial membrane1 . Autoantibodies directed against cardiolipin are part of a group known as antiphospholipid antibodies which includes anti-β2 glycoprotein 1 autoantibodies. Measurement of anticardiolipin autoantibodies is considered to be one of the most important markers to support diagnosis of antiphospholipid syndrome (APS).
APS is a systemic autoimmune disorder characterised by a combination of arterial and/or venous thromboses, pregnancy complications, such as recurrent foetal loss, together with elevated levels of antiphospholipid antibodies4 . APS was first described in patients with systemic lupus erythematosus (SLE), though it has subsequently been established that SLE may be independent of an underlying disease.
APS can occur alone – Primary APS, or in association with other conditions, such as SLE – Secondary APS6 (6). However, it has been demonstrated that anti-cardiolipin antibodies (aCLs) can be detected in patients with SLE that do not develop Secondary APS7-9. However, thromboembolic events are the most common clinical manifestation of APS
Anti-cardiolipin antibodies can recognise both cardiolipin and portions of the phospholipid-protein complex β2 glycoprotein 1-cardiolipin.
Studies have indicated an association of anti-cardiolipin IgG and IgM antibodies2,7,11-16, with thrombotic events whereas others suggest these to be linked with IgG isotype, but not IgM.

PRINCIPLE OF THE METHOD
The Anti Cardiolipin IgM assay is a two-step sandwich enzyme immunometric assay (ELISA) where patient samples, calibrators or controls are incubated on microtitre plates coated with the antigenic cardiolipin-β2 glycoprotein complex. During the incubation, antibodies present in the test sample bind to the immobilised antigen complex. After the incubation, the bound/free separation is performed by a simple solid phase washing.
A subsequent incubation occurs with anti-human IgM conjugated with horseradish peroxidase (HRP), which binds to the immobilised antibodies. A further wash step is performed to remove excess conjugate. Then, a chromogenic substrate solution containing TMB is dispensed into the wells which reacts with the conjugated HRP and a blue colour develops that changes into yellow when the Stop Solution (H2SO4) is added. The colour intensity is directly proportional to the Anti Cardiolipin IgM concentration in the sample.
Anti-cardiolipin antibody concentration in the sample is calculated through a calibration curve.

PRECAUTIONS
• Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data represented here were obtained using specific reagents listed in this Instruction For Use. 
• All reagents should be stored refrigerated at 2-8°C in their original container. Any exceptions are clearly indicated. 
• Allow all kit components and specimens to reach room temperature (22-28°C) and mix well prior to use. 
• Do not interchange kit components from different lots. The expiry date printed on box and vials labels must be observed. Do not use any kit component beyond their expiry date. 
• If you use automated equipment, the user has the responsibility to make sure that the kit has been appropriately validated for its intended use/purpose. 
• The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/or increase the background. To improve the performance of the kit on automatic systems is recommended to increase the number of washes. 
• It is important that the time of reaction in each well is held constant for reproducible results. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than 10 minutes are needed, follow the same order of dispensation. If more than one plate is used, it is recommended to repeat the dose response curve in each plate.

REAGENT STORAGE AND STABILITY
Store the kit at 2 – 8°C in the dark. 
• The kit is stable at 2 – 8°C until the expiry date stated on the external kit label. 
• Once opened, the kit is stable at 2 – 8°C for 6 months. 
• The diluted wash solution is stable for 30 days at 2-8°C. Important note: open the bag containing the Coated Microplate only when it is at room temperature and close it immediately after use.

SAMPLE COLLECTION AND STORAGE 
The assay should be performed using serum (standard sampling tubes or tubes containing serum separating gel) or plasma (lithium heparin, sodium heparin, or potassium EDTA) samples.

CALCULATION OF RESULTS 
A variety of data reduction software packages are available, which may be employed to generate the mean calibration curve and to calculate the mean concentrations of unknown samples and controls. A 4-parameter logistic (4PL) curve fit with lin-log coordinates, including Calibrator 0 is required. A smoothed spline fit including Calibrator 0 can be used. Other curve fitting algorithms are not recommended.
Alternatively, a calibration curve may be prepared on semilog graph paper by plotting mean absorbance on the Y-axis against concentration of analyte on the X-axis. Calibrator 0 should be included in the calibration curve. Read the mean absorbance value of each unknown sample off the curve.
In order for the assay results to be considered valid the kit calibrators and control must fall within the specifications detailed in the lot specific certificate of analysis. 
If a control is out of its specified range, the associated test results are invalid and samples must be retested.

QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens in each series of assays in order to check the performance of the assay. Controls should be treated as unknown samples, and the results analysed with appropriate statistical methods.
The kit controls provided in the kit should be tested as unknowns and are intended to assist in assessing the validity of results obtained with each assay plate.
The mean concentration of each control level is documented in the QC report included with each kit. These mean concentration levels are determined over several assays which are run in duplicate in multiple locations across each plate. This test is only valid if the optical density at 450 nm for the controls as well as for the calibrators (C0-C4) fall within with the respective range indicated on the Quality Control Certificate enclosed in each test kit.
DiaMetra recommends the users to maintain graphic records of the control values generated with each assay run, including the running means, SDs and %CVs. This information will facilitate the controls trending analysis relating to the performance of current and historical control lots relative to the supplied Quality Control data. The trending will assist in the identification of assays which give control values significantly different from their average range.