DiaPharma TAFI
DiaPharma TAFI, A New Two-site ELISA for Measuring TAFI
Focus on Hemostasis
Clinical Hemostasis Review
November 2002 - Volume 16 - Number 11 - Page 10
Ty Fanella, Technical Director
DiaPharma Group, Inc.
Thrombin Activatable Fibrinolysis Inhibitor (TAFI) also known as procarboxypeptidase B or procarboxypeptidase U, is a 60 kD glycoprotein synthesized in the liver, which when activated bythethrombin/thrombomodulincomplex forms activated TAFI (1). This activated form, denoted as TAFla, is formed in a stepwise manner with the generation of an intermediary protein of 56 kD and the final conversion to a 35 kD active TAFla. This activation of TAFI by thrombin provides a link between the coagulation and fibrinolytic pathways, as factors that influence the generation of thrombin will also influence the rate of clot lysis (2). TAFla has a half-life of approximately I0 minutes and is then further degraded by thrombin, trypsin, or plasmin to a 25 kD inactive fragment. TAFla is also degraded spontaneously in a temperature dependent manner which seems to be the predominant path of inactivation (3). TAFla down regulates fibrinolysis by removing C-terminal lysine and arginine residues from partially degraded fibrin. These lysine binding sites are the primary sites where plasminogen binds fibrin, and decreased plasminogen binding results in less plasmin generation by tissue-type plasminogen activator (t-PA) and impaired clot lysis (4,5).
Depending on the report, TAFI plasma concentrations vary substantially. In normals, concentrations vary from 50 nM - 275 nM (3 - 15 ug/mL). Some of these variations can be explained by the different assay methods used and in the protein concentration and mixture of the purified calibration standards. Recent reports have shown that patients taking aspirin have an impaired activation of TAFI to TAFla, therefore assays that measure TAFI levels based upon this method can show reduced values (6). Normal ranges for the concentration of TAFI in plasma are controversial. Because no reference method or international standard exists, TAFI concentrations reported in weight/volume units vary widely (7). Assay methods to measure TAFI and TAFla include both immunological and functional methods. Immunological methods can measure TAFI and TAFla as separate analytes or in combination as total TAFI in plasma. Functional methods include modified clot lysis methods and calorimetric methods.
A two-site Elisa method has been developed that measures human TAFI Antigen (Ag) in plasma and other biological fluid. All of the assay components have been optimized in order to obtain the highest sensitivity and specificity for TAFI. Both TAFI and TAFla react in a similar manner so the final measurement is for 'total" TAFI. The sample can be plasma or serum because the Sample Diluent gives similar reactivity to the various forms of TAFI in order to achieve binding equilibrium in arrange of biological fluids where TAFI measurements are of interest (Figure1). The concentration measured is similar when evaluating fresh or frozen plasmas and serum that are prepared by clotting with thrombin and calcium or after clot lysis with an excess of t-PA. These studies confirm that the reactivity of native TAFI is not influenced by pre-activated plasmas or when exposed to increased thrombolytic activities in the tested sample. This demonstrates that DiaPharma TAFI is a useful tool for investigating protein content in relationship to thrombotic risk.
The first consideration for the development of optimized ELISA tests is the choice of the antibody-pair that allows for the best capture and detection scenarios which balance incubation times and temperature with optimal sensitivity and specificity. A monoclonal antibody (murine) specific for TAFI (human) was chosen for the capture antibody. This antibody is then coated onto a Micro-ELISA plate containing 6 x 16 wells. The antibody is then stabilized and the microplate packed in a re-sealable aluminum pouch with a desiccant for moisture control. Since the assay must measure over a broad range of TAFI concentrations, a two hour incubation time was chosen because it allows maximum TAFI binding during that time period.
The second step in the DiaPharma TAFI assay procedure is the addition and incubation of the second immunoconjugated antibody. A goat polygonal antibody coupled to HRP was chosen for this antibody. The Anti-TAFI/HRP antibody forms the ELISA sandwich of monoclonal capture AB-TAFI-Anti-TAFI/ HRP immunoconjugate. The incubation time was chosen in the same manner based upon maximum binding over the range of TAFI concentrations to be measured. Three vials of TAFI Calibrator that are included in the test kit have been calibrated against two plasma pools prepared from 50 normal individuals (25 males, 25 females) and assigned a value of I 00%. Dilutions of the calibrator allow for the measurement of TAFI in the 1% - 150% concentration (2 - 200 ng/mL). This concentration was chosen to allow TAFI measurement in its median range as found in normal populations. To measure concentrations above this range, the assay is performed in the same manner but with a two-fold plasma dilution and an extension of the working range of the assay to 300% (7).
Figure 2 shows recovery studies of purified TAFI spiked in buffer and in plasma and show excellent recovery. Lower sensitivity limits were tested at the 2001 Leiden Symposium Wet Workshop, and when using TAFI immunodepleted plasmas the TAFi concentrations were found to be <1% as expected. Recent studies to measure the effect of heparin (IU/mL) to enhance fibrinolysis by inhibiting generation of TAFla, only showed slight inhibition of fibrinolysis through a heparin dependent mechanism. The data suggests that local clot bound thrombin plays a key role and the inability of heparin to inhibit clot bound thrombin has little effect on local TAFla generation (2).
The inter-assay reproducibility of the DiaPharma TAFI (For Research Use Only) kit was tested by aliquoting samples with various TAFI concentrations, freezing them at -80 °C and testing them in duplicate in 12 series. The CV% ranged from 3.8% to 8.3%. The intra-assay reproducibility was analyzed by measuring the same samples 12 times in the same series, and the CVO/o ranged from 4.26% to 5.12% (7). The appropriate quality control of the assay is insured by the inclusion of two control plasmas - one at a normal and another at a low TAFI concentration. The target values for each of the controls are lot specific and are included in the test kit with acceptable ranges. The working time for this assay is 3 hours and 5 minutes. The expiration date from manufacturing is 30 months and the assay provides excellent consistency from lot to lot. DiaPharma TAFI is the right investigative tool for measuring TAFI Ag concentrations to better understand this protein's link between the coagulation and fibrinolytic pathways.
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