DiaPharma Group, Inc.

Intended Use

The VisuLize TAFI Antigen kit is an Enzyme Immunoassay for the quantitative determination of TAFI antigen in plasma samples using an enzyme linked immuno-sorbant assay (ELISA).

Summary

TAFI (Thrombin Activatable Fibrinolysis Inhibitor), also referred to as plasma procarboxypeptidase-B, procarboxypeptidase-U and R, circulates in plasma as a zymogen with a mass of 58,000 daltons. Proteolytic activation of TAFI yields an N-terminally derived activation peptide and the C-terminal portion corresponding to the metalloprotease, activated TAFI (TAFIa). TAFIa exhibits exopeptidase activity with carboxypeptidase B-like substrate specificity capable of catalyzing the hydrolysis of C-terminal lysine and arginine residues. Cleavage of these residues on fibrin by TAFIa attenuates clot lysis by inhibiting the formation of the ternary activation complex comprising fibrin cofactor, tPA and plasminogen, thereby inhibiting plasmin generation. Although TAFI can be activated by various proteases including thrombin and plasmin, the physiological activator is proposed to be the complex thrombin-thrombomodulin since the rate of activation is stimulated 1250-fold compared to thrombin alone. However, the rate of TAFI activation is highly dependent upon its plasma concentration. Since TAFIa apparently plays a key role in connecting coagulation and fibrinolysis and significantly increases clot stability, determination of plasma concentration of TAFI is likely crucial to assess its subsequent potential antifibrinolytic effects. This has been demonstrated in a report identifying high plasma concentration of TAFI as a risk factor for thrombosis.

Principle

Strip wells are pre-coated with polyclonal antibody to human TAFI. Plasma samples are diluted and applied to the wells. The TAFI present binds to the coated antibody. After washing away unbound material, peroxidase-labeled detecting antibody is applied and allowed to bind to the captured TAFI. The wells are again washed and a solution of tetramethylbenzidine (TMB, a peroxidase substrate) is applied and allowed to react for a fixed period of time. A blue color develops which changes to yellow upon quenching the reaction with acid. The color formed is measured spectrophotometrically in a microplate reader at 450 nm. The absorbance at 450 nm is proportional to the quantity of TAFI captured onto the well. The assay is calibrated using the reference plasma provided in the kit.

Reagents -- Composition, Packaging, Storage and Stability

1. Foil pouch containing 6 strips, each containing 16 wells coated with sheep antibody to human TAFI.
2. 2 vials of Standard Reference Plasma, each lyophilized from 1 ml plasma.
3. 2 vials of Control Plasma A, each lyophilized from 1 ml plasma.
4. 2 vials of Control Plasma B, each lyophilized from 1 ml plasma.
5. 2 vials of TAFI Deficient Plasma, each lyophilized from 1 ml deficient plasma.
6. 1 vial containing 30 ml of a 10X Wash Buffer Concentrate.
7. 3 vials, each containing 20 ml of buffered Sample Diluent.
8. 1 vial of 12 ml peroxidase-labeled detecting antibody.
9. 1 vial of 12 ml of tetramethylbenzidine (TMB) substrate.
10. 1 vial of 12 ml Stop Solution containing 0.2 M Sulphuric acid.

Intact kits and un-reconstituted reagents are stable until the expiration date stated on the box and individual reagent labels when stored at 2-8°C.