TABLE 1.
Kinetic rate constants for GPIHBPl-specific mAbs by SPR
| mAb | Analyte | kon (105 M–1s–1) | koff (10−3s–1) | KD (nM) | n | Nonoverlapping Epitopes |
| RE3 | GPIHBP1 | 4.2 ± 1.8 | 1.64 ± 0.27 | 4.4 ± 1.8 | 4 | RF4 |
| RE3 | GPIHBP1-W109S | 8.2 ± 1.9 | 7.82 ± 1.80 | 10.0 ± 3.3 | 3 | |
| RE3 | GPIHBP1-Δacidic | 7.6 ± 2.7 | 1.34 ± 0.11 | 2.0 ± 0.6 | 3 | |
| RG3 | GPIHBP1 | 0.64 ± 0.22 | 0.17 ± 0.04 | 3.1 ± 1.8 | 4 | RF4 |
| RG3 | GPIHBP1-W109S | 0.93 ± 0.15 | 1.23 ± 0.06 | 13.7 ± 2.4 | 3 | |
| RG3 | GPIHBP1-Δacidic | 1.26 ± 0.21 | 0.27 ± 0.11 | 2.3 ± 1.3 | 2 | |
| RF4 | GPIHBP1 | 5.4 ± 1.9 | 0.74 ± 0.06 | 1.4 ± 0.2 | 3 | RE3, RG3 |
Kinetics rate constants were derived from data recorded with a BiacoreT200 for three-fold dilutions of various purified GPIHBP1 proteins (8) and were fitted to a 1:1 binding model. GPIHBP1 is full-length GPIHBP1; GPIHBP1-W109S contains a serine for tryptophan mutation in a highly conserved region of the Ly6 domain; GPIHBP1-Δacidic contains a deletion of the acidic domain (the first 31 amino acids of the mature protein). Data for mAbs RE3 and RG3 were processed by a multicycle protocol with mAbs that had been directly immobilized on the chip (1,000 RU), whereas the data for mAb RF4 were processed by a single-cycle protocol in which mAb RF4 was captured by an immobilized rabbit anti-mouse IgG. Epitope mapping was performed with sequential injections, as is illustrated by Fig. 3.