Cardiolipin IgG/IgA,/IgM ELISA kit

Sample

Serum or plasma

Species Reactivity

Human

Detection Method

iELISA

Intended Use

The Anti-Cardiolipin (IgAGM) ELISA test kit provides a semi-quantitative or quantitative in vitro assay for human autoantibodies of the IgAGM class against cardiolipin in serum or plasma.

Contents of Kit

1. Microplate wells, Coated with antigens: 12 microplate strips each containing 8 individual breakoff wells in a frame, ready for use --- 12 x 8. STRIPS.
2. Calibrator 1 120 RU/ml (IgAGM, human), ready for use Dark red 1 x 2.0 ml. CAL 1.
3. Calibrator 2 12 RU/ml (IgAGM, human), ready for use Red 1 x 2.0 ml. CAL 2.
4. Calibrator 3 2 RU/ml (IgAGM, human), ready for use Light red 1 x 2.0 ml. CAL 3.
5. Positive control (IgAGM, human), ready for use Blue 1 x 2.0 ml. POS CONTROL.
6. Negative control (IgAGM, human), ready for use Green 1 x 2.0 ml. NEG CONTROL.
7. Enzyme conjugate Peroxidase-labelled anti-human IgAGM (rabbit) Orange 1 x 12 ml. CONJUGATE.
8. Sample buffer ready for use Yellow 1 x 100 ml. SAMPLE BUFFER.
9. Wash buffer 10x concentrate Colorless 1 x 100 ml. WASH BUFFER 10x.
10. Chromogen/substrate solution TMB/H2O2, ready for use Colorless 1 x 12 ml. SUBSTRATE.
11. Stop solution 0.5M sulfuric acid, ready for use Colorless 1 x 12 ml. STOP SOLUTION.
12. Test instruction --- 1 booklet
13. Quality control certificate --- 1 protocol

Storage

The test kit has to be stored at a temperature between +2°C to +8°C. The calibrators and positive control must be stored in aliquots at -20°C. Unopened, all test kit components are stable until the indicated expiry date.

Performance Characteristics

Calibration: As no international reference serum exists for the polyvalent measurement of antibodies against cardiolipin, the calibration is performed in relative units (RU). However, the controls of the Anti-Cardiolipin ELISA (IgAGM) are standardized by internationally recognized standard sera.


For every group of tests performed, the extinction values of the calibrators and the relative units and/or ratio determined for the positive and negative controls must lie within the limits stated for the relevant test kit lot. A quality control certificate containing these reference values is included. If the values specified for the controls are not achieved, the test results may be inaccurate and the test should be repeated.


The binding activity of the antibodies and the activity of the enzyme used are temperature-dependent. It is therefore recommended using a thermostat in all three incubation steps. The higher the room temperature (+18°C to +25°C) during the incubation steps, the greater will be the extinction values. Corresponding variations apply also to the incubation times. However, the calibrators are subject to the same influences, with the result that such variations will be largely compensated in the calculation of the result.

Antigen: Cardiolipin is a negatively charged phospholipid which is located in high concentrations in the inner mitochondrial membrane (of nine known mitochondrial antigen types it is classified as M1). Phospholipids consist of a phosphoric acid esterified to, on the one hand, a glycerol derivative, and on the other hand, serine, choline, ethanolamine, inositol or glycerol. The glycerol derivative contains two fatty acids with double bindings of varying number and length. The glycerol derivative together with the esterified phosphoric acid form a phosphatidic acid. In cardiolipin two phosphatidic acids are linked to a further glycerol.

Antibodies against cardiolipin are probably a subtype of a class of closely related antibodies to anionic phospholipids (e.g., cardiolipin, phosphatidylserine, phosphatidylinositol) which differ in their affinities. A subpopulation of antibodies against cardiolipin (ca. 75%) relies on a plasma protein (β2- glycoprotein I, GPI) as a cofactor for antigen recognition. It is presently not clear whether this antibody population only recognizes epitopes of the GPI or also epitopes of the cardiolipin. GPI is known to interact only with anionic but not with neutral phospholipids (e.g. phosphatidyethanolamine).

Detection Limit

1.2 RU/mL

General Notes

The Anti-Cardiolipin (IgAGM) ELISA test kit provides a semi-quantitative or quantitative in vitro assay for human autoantibodies of the IgAGM class against cardiolipin in serum or plasma for the diagnosis of anti-phospholipid syndrome (APS).