Fig 1.
Calculating the concentration of active scFv.
The scFv (142 nM, as determined by measuring the optical density at 280nm) was injected over a chip surface with high ligand immobilisation at several flow rates. (a) The association phase of the sensorgrams was divided into six response intervals (I-VI). (b) Comparison of the experimental (dR/dtreg) and calculated (dR/dtfit) binding rates for each response interval. (c) Lr (red) and [Abulk] (blue) were calculated by global fitting of the data at each flow rate using Eq 2, using dR/dtreg as slopes for each response interval. Lr values decrease with the increase of the response interval, while the calculated [Abulk] values are contained in a small interval (30.622–32.098 nM). The calculated concentration of active scFv is the average [Abulk] from each response interval: 30.97 ± 0.559 nM (SD = 1.8%).
Fig 2.
Kinetic measurements on surfaces with different ligand densities.
Six concentrations of scFv (2-fold dilutions from 24 to 0.75 nM) were injected at 25°C. The responses from the in-line reference flow cell and blank injections were subtracted from all curves. The curves were globally fitted to a 1:1 Langmuir binding model. For each surface density, the experimental sensorgrams (coloured lines) were overlain with the theoretical fitted curves (black lines). In each case, the relative residual plots show the difference between the experimental and the theoretical curves expressed as a percentage of the observed response for each analyte concentration. The spikes observed at the beginning and the end of analyte injections are the result of in-line reference subtraction with the slight sensorgran misalignment introduced with sequential analyte flow in the multichannel mode. Nevertheless, residuals are mostly within 1% of Robs. The surface density, expressed as the maximum analyte binding capacity (Rmax) was determined experimentally: Rmax ≈ 49 RU (a); 465 RU (b); 3100 RU (c).
Table 1.
Comparing values of affinity constant determined by surface (kinetic and equilibrium) and solution (equilibrium) assays for the scFv.
Table 2.
Kinetic and affinity constants for 15-I-C5.
Fig 3.
Monoclonal 15-I-C5 (5 nM) was mixed with different concentrations of (+)-ABA (0.5–500 nM) for 1 hour at 25°C. The equilibrium mixtures were injected over a sensor chip surface with high b-PEG-ABA density and the initial binding slopes were measured for each sample. The values were plotted against the concentration of free ABA and the data points were fitted to a 4-parameter equation. KD was estimated from the corrected inflection midpoint of the fitted curve (Stöcklein et al., 1998). The affinity in solution of 15-I-C5 for (+)-ABA was estimated to 1.13 nM.
Fig 4.
Cross-reactivity of the anti-ABA-scFv.
MBP-antiABA-scFv (25 nM) was mixed with different concentrations of each test compound for 1 hour at 25°C. The concentration of free scFv in the equilibrium mixtures was plotted against the concentration of test compound. The structures of each competitor are shown framed in the same colours as the data in the dose graph. There is no box for the racemic mix. The colour code for each compound is shown on the left of the plot.
Table 3.
Selectivity of MBP-aABA-scFv.