Table 1.
rHBcAgd and rHBeAgd immune complexes: molecular weights and binding affinities. See also Figure S2, Figure S3 and Figure S4.
| Immune Complex a | Mw (AUC) b kDa | Mw (Sequence) c kDa | Binding affinity d Kd (M) |
|---|---|---|---|
| rHBcAgd-Fab e13 | 117.1 (±1.5) | 128.66 | 3.4 ×10−12 |
| rHBcAgd-scFv e13 | 80.0 (±1.3) | 86.92 | nd |
| rHBeAgd-Fab e13 | 127.00 (±1.5) | 130.87 | 3.1 ×10−11 |
| rHBeAgd-scFv e13 | 84.51 (±1.4) | 89.20 | nd |
| rHBeAgd-Fab e13-scFv me6 | 182.5 (±3.5) | 187.50 | nd |
| rHBcAgd-Fab e21 | 126.6 (±1.1) | 128.44 | 2.0 × 10−12 |
| rHBeAgd-Fab e21 e | 136.41 (±2.5) | 130.73 | 1.1 × 10−10 |
| rHBcAgd-Fab me6 | 138.52 (±2.8) | 133.11 | 2.7 [2.0] d × 10−9 |
| rHBcAgd-scFv me6 | 93.7 (±1.4) | 90.11 | [1.1] × 10−9 |
| rHBeAgd-Fab me6 | 138.86 (±3.0) | 135.2 | 1.1 × 10−8 |
| rHBeAgd-scFv me6 | 99.4 (±1.3) | 92.21 | 1.6 × 10−9 |
Chimeric antibody fragments: e13 and e21 (rabbit/human); me6 (murine/human).
Molecular weights were determined by sedimentation equilibrium. The equilibrium profiles for scFv e13 and Fab e21 bound to rHBcAgd and rHBeAgd are shown in Figure S3.
Molecular weights were calculated from the amino acid sequences for a 1:2 complex (i.e. 1 rHBc/eAgd plus 2 antibody fragments). In all cases, there is a close match between the experimental and expected molecular weights, indicating that the binding stoichiometry is 1:2. The mass of the rHBeAgd-Fab e13-scFv me6 complex corresponds to a stoichiometry of 1:2:2 (i.e. 1 rHBeAgd plus 2 Fab plus 2 scFv), indicating that Fab e13 and scFv me6 have non-overlapping epitopes.
Binding affinities were determined by surface plasmon resonance (Biacore) with the antibody fragments immobilized (ligands) and the HBV proteins (analytes) titrated. The two values in brackets were determined with the HBV proteins immobilized and the antibody fragments titrated.
Binding stoichiometry for the rHBeAgd-Fab e21 complex was previously reported to be 1:05 [12], compared to 1:2 in the current study. This discrepancy is due to incomplete saturation of the rHBeAgd with Fab e21 in the earlier study. The binding affinity remains the same.