Table 1.
Apparent kinetic and equilibrium constants derived from binding of gp120 to CD4i antibodies in biosensor experiments
| Antibody | kon, 1/Ms | koff, 1/s | Kd, nM |
|---|---|---|---|
| Binding to 17B | |||
| 8x | 1 × 105 | 2 × 10−3 | 15.0 |
| 8x/CD4 | 2 × 105 | 9 × 10−4 | 4.5 |
| IIIB | 8 × 103 | 2 × 10−5 | 2.5 |
| IIIB/CD4 | 3 × 105 | 5 × 10−5 | 0.2 |
| Binding to 48d | |||
| 8x | 2 × 105 | 1 × 10−3 | 6.0 |
| 8x/CD4 | 3 × 105 | 6 × 10−4 | 2.0 |
| IIIB | 1 × 104 | 5 × 10−5 | 5.0 |
| IIIB/CD4 | 5 × 105 | 3 × 10−5 | 0.1 |
The CD4i antibodies 17b and 48d were attached to the biosensor surface, and serial dilutions of 8x and IIIB gp120 were measured for both binding and dissociation. Similar measurements were also performed with serial dilutions of Env that had been premixed with a saturating amount of sCD4. All binding reactions were performed at 25°C. A sample sensogram is shown in Fig. 4. The best fitted values for the slopes of the linearized plots of the data (r2 ≥ 0.98) are reported. The parameters estimated by fitting the simple 1:1 Langmuir interaction model globally were within 15% of the reported values. The off rates for IIIB and IIIB/sCD4 for both antibodies were so slow that they were at the limits of detection for the biosensor, making the affinity constants derived from these values less accurate.