Two-site immuno-assay research ELISA method for the measurement of human Beta 2 Glyocoprotein I (Beta2GPI) on plasma or any biological sample where it must be measured. The assay is designed with two murine monoclonal antibodies specific for Beta2GPI, the first one for coating the microplate (immunocapture) and the second one, labeled with horseradish peroxidase (HRP), for revelation. This assay measures homogeneously B2GPI in plasma.
Assay Principle
In a first step, the diluted tested plasma or biological fluid is introduced into a microwell coated with highly purified polyclonal antibodies specific for human Beta2GPI. When present, this protein is captured onto the solid phase. Following a washing step, the immunoconjugate, which is a polyclonal antibody coupled to horse radish peroxidase (HRP), is introduced, and binds to other free epitopes of immobilized Beta2GPI. Following a new washing step, the peroxidase substrate, Tetramethylbenzidine (TMB) in presence of hydrogen peroxide (H2O2), is introduced and a blue colour develops. When the reaction is stopped with Sulfuric Acid, a yellow color is obtained. The amount of color developed is directly proportional to the concentration of human Beta2GPI:Ag in the tested sample.
Test Sample
- Trisodium Citrate or Na2 EDTA anticoagulated human plasma.
- The assay can be performed on serum.
- Any biological fluid where Beta2GPI:Ag must be measured.
Reagents -- Preparation, Storage & Stability
- Micro ELISA plate: 12 strips x 8 wells, coated with highly purified polyclonal antibodies specific for human Beta2GPI, then stabilised; the plate is packed in an aluminium pouch hermetically sealed in presence of a desiccant. Open the plastic pouch and take off the required amounts of 8 well strips for the test series. When out of the pouch, the strips must be used within 30 minutes. Unused strips can be stored at 2-8°C for 4 weeks in their original aluminium pouch, in presence of the desiccant, hermetically closed and protected from any moisture, and stored in the provided microplate storage bag (minigrip).
- Beta2GPI Sample Diluent: 2 vials x 60ml, ready to use. When open, it can be used for 4 weeks, stored at 2-8°C. Reagent contains 0.05% Kathon CG.
- Beta2GPI Calibrator: 3 vials, lyophilised. When restored with 2 ml of Beta2GPI - Sample Diluent, a solution containing "C" ng/ml of human Beta2GPI is obtained. This solution is stable for at least 8 hours at room temperature (18-25°C) or 72 hours at 2-8°C.
- Beta2GPI Control I (high): 1 vial x 1 ml, lyophilised. Restore with 1 ml of Beta2GPI-Sample Diluent.
- Beta2GPI Control II (low): 1 vial x 1 ml, lyophilised. Restore with 1 ml of Beta2GPI-Sample Diluent.
- Anti-(h)-Beta2GPI-HRP immunoconjugate: 1 vial x 25 ml, ready to use. Each vial must be restored with 7.5 ml of Beta2GPI Conjugate Diluent. Let the pellet to be completely dissolved before use, and shake the vial gently in order to homogeneize the content. The restored conjugate is stable for at least 24 hours at room temperature or for at least 4 weeks at 2-8°C.
- Beta2GPI Conjugate Diluent: It is ready to use. When open, it can be used for 4 weeks, stored at 2-8°C. Reagent contains 0.05% Kathon CG.
- Wash Solution: 1 vial x 50 ml x 20 fold concentrated Wash Solution. Incubate the vial for 15-30 minutes in a water bath at 37°C until complete dissolution of solids, when present. Shake the vial and dilute the amount required 1:20 in distilled water (the 50 ml contained in the vial allow preparing 1 liter of Wash Solution). The Wash Solution must be stored at 2-8°C in its original vial and used within 4 weeks following opening. The diluted Wash Solution must be used within 7 days, when protected from any contamination and stored at 2-8°C. This reagent contains 0.05% Kathon CG.
- TMB substrate: 1 vial x 25 ml peroxidase substrate, ready to use: 3,3',5,5' -Tetramethylbenzidine: containing hydrogen peroxide. When open, it can be used for 4 weeks, stored at 2-8°C.
- Stop solution: 1 vial x 6 ml of 0.45M Sulfuric acid (Stop solution), ready to use.
Note: a minimum order of 20 kits is required for this item.