Table 1.
Analytical techniques used to characterize protein therapeutics.
Figure 1.
ELISA based binding assays using aptamers as surrogate antibodies.
(A & B) Illustrations showing assay platforms using either polyclonal antibody (A) or aptamers (B) to detect thrombin. Both assays use an identical anti-thrombin monolonal antibody as the capture antibody and identical reagents for detection (NutrAvidin-HRP and TMB). Assays used in Fig. 6 however use a polyclonal antibody as the capture antibody, one of four monoclonal antibodies as detectors and anti-mouse-HRP as a secondary antibody (C) ELISA assay showing the limit of detection for purified human thrombin using the antibody based assay illustrated in (A). (D) ELISA assay showing the limit of detection for purified human thrombin using the aptamer based assay illustrated in (B).
Figure 2.
Affinities of the six aptamers used in this study determined using the aptamer based ELISA.
A fixed concentration of thrombin (Evithrom), 150 nM was captured by the anti-thrombin monoclonal antibody, 5 µg/ml. Binding of increasing concentrations of the six anti-thrombin aptamers (A, Aptamer 30-14, B, Aptamer 30-8, C, Aptamer 60-18/38, D, Aptamer 30-16/27, E, Aptamer 30-38/27 and F, Aptamer G15D) was determined using the assay depicted in Fig. 1B. Data presented shows the mean (± SD) of three experiments where the aptamers were generated independently for each experiment.
Table 2.
Sequences of aptamers used in this study.
Table 3.
Kinetic parameters for the binding of aptamers to Recothrom.
Figure 3.
Comparing three topical thrombin products using binding assays with aptamers as detector reagents in an ELISA format.
(A) The three topical thrombin products were electrophoresed on a 12% NuPAGE gel and stained using Colloidal Blue®. The thrombin and HSA (used as an exipient) bands are indicated with red and blue arrows respectively. (B) Kinetics of binding of the Aptamer 30-8 to the purified human thrombin products Recothrom (squares) and Evithrom (circles). (C) The affinities (apparent Kds) of all six aptamers to both Recothrom and Evithrom were determined as shown in (B). The apparent Kd values for Recothrom (X-axis) were plotted against those for Evithrom (Y-axis) and were shown to be highly correlated (r = 0.996, P<0.05).
Figure 4.
Conformational changes in thrombin following heat treatment in the presence of 60% sucrose.
The two purified human topical thrombin products were heated for up to 17 h at 60°C in the presence of 60% sucrose in duplicate. (A) The catalytic activity of the Recothrom (clear bars) and Evithrom (grey, filled bars) samples was determined at time 0 and 17 h; the mean activity (± SD) of three independently treated samples is depicted. (B) Following heat treatment both samples were electrophoresed on a 12% NuPAGE gel and stained using Colloidal Blue®. As a control (Con) the thrombin samples were heated at 60°C in PBS the absence of sucrose. (C) The binding kinetics of the heat-treated thrombin at time 0 h (Blue open circles) and 17 h (purple open triangles) were determined using the antibody based ELISA illustrated in Fig. 1A. Samples heated in the presence of PBS alone are shown as red filled circles. (D) The binding kinetics of the heat-treated thrombin at time 0 h (Blue open circles), 1 h (green open squares) and 17 h (purple open triangles) were determined using the aptamer based ELISA illustrated in Fig. 1B. Samples heated in the presence of PBS alone are shown as red filled circles.
Figure 5.
Heat treatment of human thrombins in the absence and presence of 60% sucrose.
The two purified human topical thrombin products were heat-treated as described in Fig. 3. Control samples were heated in for 17 h at 60°C in PBS (in the absence of 60% sucrose). Binding of either the antibody or each of the six aptamers was monitored to (A) Recothrom or (B) Evithrom at time 0 and 17 h. The percent of the binding at time 0 (mean ± SD, n = 3) is represented. The aptamers used are shown on the figure.
Figure 6.
Effect of heat treatment of thrombin in the presence of 60% sucrose monitored using anti-thrombin monoclonal antibodies.
The binding kinetics of the heat-treated thrombin (Recothrom) at time 0 h (Green open squares) and 17 h (purple open triangles) were determined using an antibody based ELISA. The polyclonal anti-thrombin antibody, PAHT-S-B was used as the capture antibody and a panel of monoclonal antibodies as detector antibodies as described in the Materials & Methods. The binding kinetics of the mouse anti-thrombin antibodies EST-2 (A), EST-4 (B), sc-59717 (C) and AH-5020 (D) are depicted.
Figure 7.
Estimating the binding of aptamers to thrombin in a label free assay.
The binding of aptamers to Recothrom was monitored in real time using BLI and the kinetic parameters obtained using the Octet data analysis software version 7.0. (A) Correlation between Kdis values obtained in the label-free assay with the apparent Kds estimated using the ELISA based assay (r = 0.842, P<0.05). (B) Effect of heat-treatment of human thrombins on the kdis for all 6 aptamers. Recothrom was heat-treated as described in Fig. 3 and the kdis determined following heat treatment for 0 and 17 h. The percent of the binding at time 0 is represented. Data presented is the mean (± SD) of three independent experiments.