Factor IX (F.IX, Christmas Factor) is a vitamin K-dependent glycoprotein produced in the liver. Plasma concentration of F.IX is normally around 5ug/ml (87nM) in plasma. The biological importance of F.IX is demonstrated in Haemophilia B (Christmas disease), an X-linked congenital bleeding disease resulting from a quantitative (low activity and low antigen) or qualitative (low activity and normal antigen) defect in F.IX function. In its zymogen form F.IX is a single chain molecule of 55kD. It contains two EGF-like domains and an amino-terminal domain containing 12-gamma-carboxy-glutamic acid (Gla) residues. These Gla residues allow F.IX to bind divalent metal ions and participate in calcium-dependent binding interactions. The activation of F.IX occurs by limited proteolysis in the presence of calcium by activated factor XI (FXIa) and/or by a complex of VIIa/tissue factor/phospholipid and activated Factor X between residues Arg146- Ala147 and between Arg180-Val181. The terminal activated product in either case is F.IXa-beta, a two-chain enzyme consisting of a heavy chain (28kD), a light chain (18D) and an activation peptide product of 11kD. F.IX can also be cleaved into inactive products by thrombin and by elastase. The activity of F.IXa-beta in plasma is inhibited by antithrombin and
Principle of Sandwich ELISA
Affinity-purified antibody to F.IX is coated onto the wells of a microtitre plate. The plates are washed and plasma or other fluids containing F.IX are applied. The coated antibody will capture the F.IX in the sample. After washing the plate to remove unbound material, a peroxidase conjugated second antibody to F.IX is added to the plate to bind to the captured F.IX. After washing the plate to remove unbound conjugated antibody, the peroxidase activity is expressed by incubation with o-phenylenediamine (OPD). After a fixed development time the reaction is quenched with the addition of H2SO4 and the color produced is quantified using a microplate reader. The color generated is proportional to the concentration of F.IX present in the sample.
Kit Components
F0017-09A: Capture Antibody, 1x500ul. Supplied as a liquid in 50% glycerol. Affinity purified F.IX Pab for coating ELISA plates. F0017-09B: Dection Antibody, 1x500ul. Supplied as a liquid in 50% glycerol. Affinity purified F.IX Pab (HRP) for detection of captured F.IX.
Storage and Stability
May be stored at 4°C for short-term only. For long-term storage, aliquot and store at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Materials required but not supplied
Coating Buffer: 50mM Carbonate, To prepare 1 liter; 1.59g of Na2CO3 and 2.93g of NaHCO3. Adjust pH to 9.6. Store at 4°C up to 1month.
PBS (base for wash buffer): To prepare 1 liter; 8.0g NaCl, 1.15g Na2HPO4, 0.2g KH2PO4, 0.2g KCl, and 1.0ml of Tween-20, up to 1 litre. Adjust pH to 7.4. Store at 4°C up to 1month; discard if there is evidence of microbial growth.
Sample Diluent: HBS-BSA-EDTA-Tween, 5.95g HEPES, 1.46g NaCl, 0.93g Na2EDTA, 2.5g Bovine Serum Albumin (RIA grade), dissolved in 250ml water. Add 0.25ml Tween-20. Adjust pH to 7.2 with NaOH, then make up to a final volume of 250ml with water. Aliquot and store frozen at -20°C.
Wash Buffe: PBS-Tween (0.1% v/v); To 1 liter of PBS, add 1ml of Tween-20. Check that pH is 7.4. Store at 4°C up to 1 week.
Substrate Buffer: Citrate-Phosphate buffer pH 5.0. 2.6g Citric acid and 6.9g Na2HPO4. Make up to final volume of 500ml with water. Store at 4°C up to 1month.
OPD (O-Phenylenediamine), 5mg tablets. Make up immediately before use. Dissolve 5mg OPD in 12ml substrate buffer then add 12ul 30% H2O2. Note -Toxic.
Stop Solution: 2.5M H2SO4, Caution; Very corrosive. Generates heat on dilution. Where stock sulfuric acid is 18M, add 13.9ml to 86ml H2O. Store at room temperature. EIA-grade microplates (Dynatech Immulon-4), microplate washer, and microplate reader.
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