DiaPharma Group, Inc.

DiaPharma Factor XIII-A kit is a research use only, one step "two site" immuno-assay for measuring human Factor XIII (subunit A) (FXIII-A) in plasma, or in any fluid where FXIII can be present.

ASSAY PRINCIPLE

First, the immunoconjugate, which is a polyclonal antibody specific for FXIII-A coupled to horse radish peroxidase (HRP), is introduced into the microwells coated with a polyclonal antibody specific for FXIII-A. Then, the diluted tested sample is immediately introduced, and the immunological reaction starts. When present, FXIII-A binds onto the polyclonal antibody coated solid phase through one epitope, and fixes the polyclonal antibody coupled to HRP through free epitopes. Following a washing step, the peroxidase substrate, 3,3',5,5' - Tetramethylbenzidine (TMB), in presence of hydrogen peroxide (H2O2), is introduced and a blue color 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 FXIII-A in the tested sample.

TEST SAMPLE

• Trisodium Citrate anticoagulated human plasma.

REAGENTS PREPARATION, STORAGE AND STABILITY

1. COAT: Micro ELISA plate, containing 12 strips of 8 wells, coated with a sheep polyclonal antibody specific for human FXIII-A, 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 amount 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).
2. Sample Diluent (SD): 2 vials containing 50ml, ready to use. When open, it can be used for 4 weeks, stored at 2-8°C, and provided that any bacterial contamination is avoided during use. This reagent contains 0.05% Kathon CG.
3. Factor XIII Calibrator (Cal): 3 vials, lyophilised. When restored with 2 ml of Sample Diluent, a plasma containing a concentration "C" (expressed in %) of human FXIII-A is obtained. This concentration (in the range 120-200% according to the lot), established by reference to a normal citrated human plasma pool, is accurately determined for each lot. The exact concentration is indicated on the flyer provided with each kit. Restore each vial with 2 ml of Sample Diluent in order to obtain a plasma containing a FXIII-A concentration "C", already diluted 1:50 (fifty fold). This solution is stable for at least 8 hours at room temperature.
4. Plasma FXIII Control I High (CI): 1 vial containing 0.5 ml of lyophilized (human plasma). Restore with 0.5 ml distilled water.
5. Plasma FXIII Control II Low (CII): 1 vial containing 0.5 ml of lyophilized (human plasma). Restore with 0.5 ml distilled water.
   Note: The FXIII-A concentrations and acceptancy ranges for controls can vary from lot to lot, and are indicated on the flyer provided in the kit.
6. Anti-(h)-FXIII-HRP immunoconjugate (IC): 3 vials of a polyclonal antibody coupled to HRP, lyophilised.
7. Conjugate Diluent (CD): 1 vial of 25 ml, ready to use.
8. Wash Solution (WS): 1 vial of 50 ml of 20 fold concentrated. 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.
9. TMB: 1 vial of 25 ml peroxidase substrate: 3,3',5,5' - Tetramethylbenzidine containing hydrogen peroxide. Ready to use.
10. Sulfuric acid (SA): 1 vial of 6 ml of 0.45M (Stop solution). Ready to use.
    Note: Use only components from a same kit lot. Do not mix components from different lots, when running the assay. When open, it can be used for 4 weeks, stored at 2-8°C, and provided that any bacterial contamination is avoided during use.

In their original packaging box, before use, when stored at 2-8°C, the unopened reagents are stable until the expiration date printed on the box.

ASSAY PROCEDURE

Remove the required number of strips from the aluminium pouch, for the series of measures to be performed. Then put the strips in the frame provided. In the different wells of the micro ELISA plate introduce the reagents and perform the various assay steps as indicated on the following table:

Minimum order of 20 kits required