REAADS® Anti-Beta2 Glycoprotein I (Aβ2GPI) IgM

The REAADS® Anti-Beta2 Glycoprotein I (Aβ₂GPI) IgM test kit is an indirect enzyme-linked immunosorbent assay (ELISA) for the semi-quantitative determination of IgM anti-β₂GP1 antibodies in human serum or citrated plasma (3.2% sodium citrate). A single point or multi point calibrator is used to measure IgM anti-β₂GP1 antibody concentrations in M units. For the detection of IgM anti-β₂GPl antibodies in individuals with systemic lupus erythematosus (SLE) and lupus-like disorders (anti-phospholipid syndrome).

Reagents

• 12 x 8 stabilized β₂GPl (from human serum) coated microwells with frame
• 60 ml Sample Diluent IV (blue-green solution)
• 3 vials (0.250 ml) IgM β₂GPl Calibrator Serum* (1-high, 2-moderate, 3-low) (human); see vial label for antibody concentration in M units. Calibrator 2 should be used when performing single point calibration
• 0.250 ml IgM β₂GPl Positive Control Serum* (human); see vial label for expected M unit range
• 0.250 ml Normal Control Serum* (human); see vial label for expected M unit range
• 15 ml anti-human IgM (goat) HRP-Conjugated Antibody Solution (red solution)
• 15 ml One Component Substrate Solution (TMB and H2O2); ready to use
• 15 ml Stopping Solution (0.36 N sulfuric acid)
• 2 bottles (30 ml) Wash Concentrate (33X PBS/Tween)

Store at 2 – 8°C. Do Not Freeze.

Materials Required but not Supplied

• Reagent grade water to prepare PBS wash solution and to zero or blank the plate reader during the final assay step
• Graduated cylinders
• Precision pipettors capable of delivering between 5 µL and 1000 µl, with appropriate tips
• Miscellaneous glassware appropriate for handling small volumes
• Flasks or bottles, 1 liter
• Wash bottles, preferably with the tip partially cut back to provide a wide stream, or an automated or semi-automated washing system
• Disposable gloves
• Plate-reading spectrophotometer capable of reading absorbance at 450 nm (650 nm reference if dual beam)
• Multichannel pipettors capable of delivering to 8 wells simultaneously
• Microdilution tubes and a 96-well microdilution tube holder for sample dilutions and rapid delivery to microwell plate

Advanatages

• Excellent clinical correlation
• Color coded reagents
• Total incubation time: 40 minutes
• Convenient, cost effective
• Choice of single or multi-point calibration

Measurement Principle

The REAADS® IgM anti-β₂GPI test kits are indirect enzyme linked immunosorbent assays for the semiquantitative determination of anti-β₂GP1 antibodies in human serum. A single point or multi point calibrator is used to measure IgM anti-β₂GP1 antibody concentrations in M units.

The test is performed as an indirect ELISA. Diluted serum or plasma samples, calibrator sera, and controls are incubated in microwells coated with purified human β₂GPl. Incubation allows the anti-β₂GPl antibodies present in the samples to react with the immobilized antigen. After the removal of unbound serum or plasma proteins by washing, antibodies specific for human IgM, labeled with horseradish peroxidase (HRP), are added forming complexes with the β₂GPl bound antibodies. Following another washing step, the bound enzyme-antibody conjugate is assayed by the addition of a single solution containing tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) as the chromogenic substrate. Color develops in the wells at an intensity proportional to the serum concentration of anti-β₂GPl antibodies.

Results are obtained by reading the O.D. (optical density or absorbance) of each well in a spectrophotometer. Calibrator sera are provided, with the IgM anti-β₂GPl antibody concentrations expressed in M 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 sera by the O.D. value of the calibrator provides a conversion factor. The O.D. values of the other samples are multiplied by the conversion factor to obtain IgM anti-β₂GPl antibody concentrations in M units. For multipoint calibration, perform a linear regression analysis with calibrator values against calibrator O.D.s. Controls and patient results are determined from the calibration curve.

Assay Procedure

1. The assay can be performed with a single point calibration (Calibrator 2) or a four-point calibration curve (Calibrators 1, 2, and 3 plus sample diluent/reagent blank as Calibrator 4 equal to 0 M units). A reagent blank control should also be run with the single point and multipoint calibration method. Sample Diluent without serum or plasma is added to the well. This well will be treated the same as a control or patient sample in subsequent assay steps.
2. Remove any microwell strips that will not be used from the frame and store them in the bag provided.
3. Prepare a 1:50 dilution of the calibrators, controls, and patient samples in sample diluent (blue-green solution); e.g., 10 µl sample added to 490 µl Sample Diluent equals a 1:50 sample dilution.
4. Add 100 µl of diluted calibrators (including the reagent blank/Calibrator 4), controls, and patient samples to the appropriate microwells.
5. Incubate 15 minutes at room temperature. After the incubation is complete, carefully invert the microwells and empty the sample fluid. Do not allow samples to contaminate other microwells.
6. Wash 4 times with wash solution. Each well should be filled with wash solution per wash. Invert microwells between each wash to empty fluid. Use a snapping motion of the wrist to shake the liquid from the wells. To retain microwell modules during washing, the frame must be squeezed at the top and bottom of the longest sides. Blot on absorbent paper to remove residual wash fluid. Do not allow wells to dry out between steps.
7. Add 100 µl anti-human IgM HRP-Conjugated Antibody Solution (red) to the wells.
8. Incubate for 15 minutes at room temperature. After the incubation is complete, carefully invert the microwells and empty the conjugate solution.
9. Wash 4 times with wash solution, as in step 6. Use a snapping motion to drain the liquid and blot on absorbent towels after the final wash. Do not allow the wells to dry out.
10. Add 100 µl One Component Substrate to each well and incubate for 10 minutes at room temperature. Add substrate to the wells at a steady rate. Blue color will develop in wells with positive samples.
11. Add 100 µl Stopping Solution (0.36 N sulfuric acid) to each well to stop the enzyme reaction. Be sure to add the acid to the wells in the same order and at the same rate as the Substrate was added. Blue Substrate will turn yellow and colorless solution will remain colorless. Blank or zero the plate reader against an air or a water blank well. Read the O.D. of each well at 450 nm (and 650 nm reference if dual beam). The O.D. values should be measured within 5 minutes of the addition of the Stopping Solution.

Clinical Performance

Clinical Specificity: serum samples from multiple healthy blood donor populations were assayed for the presence of IgG, IgM, IgA anti-B2GPI antibodies. Specificity was shown to be 100%, 93%, and 96%, respectively for the three isotypes.

Clinical Sensitivity: an unselected SLE population was tested to determine the clinical sensitivity of the anti-B2GPI assays.

Sensitivity was 23% for IgG, 20% for IgM and 25% for IgA. The clinical sensitivity of the assay for thrombosis was determined by comparing anti-B2GPI 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 (control). The results are shown below:

Background

Anti-phospholipid antibodies are a heterogeneous group of immunoglobulins that bind to several anionic phospholipids, including cardiolipin and phosphatidylserine. High serum levels of anti-phospholipid antibodies are frequently detected in patients with autoimmune (e.g., SLE) and non-autoimmune diseases, as well as in apparently healthy individuals. These antibodies have been associated with an increased risk for recurrent arterial and venous thrombotic events, thrombocytopenia, and fetal loss. These manifestations are the main features of the anti-phospholipid syndrome (APS).

Most autoimmune anti-phospholipid antibodies require a serum cofactor (β₂GPl) for optimal binding. It has been shown that many anti-phospholipid antibodies may react to a neoepitope formed on the β₂GPl molecule by the interaction between the phospholipid and β₂GPl. Most assays for anti-phospholipid antibodies contain bovine serum as the source of cofactor. More recently, it has been shown that the binding of β₂GPl to the microwell surface may produce a neoepitope similar to that when combined with a phospholipid and the results with this system showed a good correlation with the anti-phospholipid syndrome. The serologic detection of anti-β₂GPl antibodies provides enhanced clinical sensitivity for thrombosis. The REAADS® Anti-β₂GPl ELISA test kit uses the well known ELISA format to detect anti-β₂GPl antibodies in human serum.

Patients with positive reactions to both anti-phospholipid and anti-β₂GPl assays were more likely to have clinical complications than those positive for only one. Higher prevalence and mean serum levels of IgM anti-β₂GPl antibodies have been reported in autoimmune patients. In addition, anti-β₂GPl antibodies in SLE patients correlated with clinical manifestations of anti-phospholipid syndrome.