TABLE 1.
Inhibition of BC7 binding to NHBE cells by BEC and epidermal cytokeratins and polyclonal antibody to epidermal cytokeratinsa
| Inhibitor(s) | CK13 content (μg/ml) | % Inhibition (range) |
|---|---|---|
| None (control) | 0.0 | |
| Mixed epidermal cytokeratins (μg/ml) | ||
| 2.5 | 0.025 | 5.2 (2.2–8.7) |
| 5.0 | 0.05 | 9.4 (6.3–13.1) |
| 10.0 | 0.1 | 32.3 (28–40) |
| 20.0 | 0.2 | 58.6 (51–67) |
| BEC CS fraction (cell number) | ||
| 104 | 0.08 | 37 (32–42) |
| 105 | 0.84 | 57 (49–63) |
| 106 | 8.4 | 73 (72–75) |
| Planar stratum corneum cytokeratins (20 μg/ml) | 0.0 | 0.0 |
| Polyclonal antibody to mixed epidermal cytokeratins (dilution) | ||
| 1:250 | 58 (54–61) | |
| 1:100 | 68 (65–69) | |
| 1:50 | 72 (69–73) |
NHBE cells were grown in 96-well plates until they were 80 to 90% confluent. Wells were blocked with 3% BSA for 1 h at 37°C, washed with PBS, and then incubated for 1 h at 37°C with BC7 (107 CFU/ml) preincubated with potential inhibitors (or PBS to provide background binding data). When the antibody to mixed epidermal cytokeratins was used as an inhibitor, NHBE cells were first incubated with the antibody for 1 h and then washed to remove excess antibody prior to the addition of bacteria. Wells were washed five times with PBS to remove nonbound bacteria and the bound bacteria quantitated by enzyme-linked immunosorbent assay by using an antibody specific to B. cepacia. Values represent the average of triplicate experiments, and the numbers in parentheses are the total ranges of values obtained.