Table 1.
OPKA protocols used in this study
| OPKA typea | Type of assay | Colony enumeration method | No. of 3-fold serum dilutions tested | Assay buffer | E/Tb | Opsonizationc | Phagocytosisd | Reference |
|---|---|---|---|---|---|---|---|---|
| 1A | Endpoint dilution killing 50% of target bacteria | Automated counter | 8 | HBSS + 0.1% gelatin + 5% FBS | 400:1 | (Test serum + target bacteria) × 30 min, RT with shaking | (12.5% BRC + HL-60) × 45 min, 37°C, 5% CO2 with shaking | Modified from Burton and Nahm (38) |
| 1B | Endpoint dilution killing 50% of target bacteria | Automated counter | 8 | HBSS + 0.1% gelatin + 5% FBS | 400:1 | (Test serum + target bacteria) × 30 min, RT with shaking | 12.5% BRC × 30 min, 37°C, 5% CO2 with shaking; HL-60, × 45 min, 37°C, 5% CO2 with shaking | This study |
| 1C | Endpoint dilution killing 50% of target bacteria | Automated counter | 8 | HBSS + 0.1% gelatin + 5% FBS | 400:1 | (Test serum + target bacteria) × 30 min, RT with shaking | (12.5% BRC + HL-60) × 75 min, 37°C, 5% CO2 with shaking | This study |
| 2A | % killing of bacteria at indicated serum dilutions | Manual | 1–4 | HBSS + 0.1% gelatin + 5% FBS | 200:1 | (Test serum + target bacteria) × 30 min, 37°C, 5% CO2 with shaking | (7% BRC + HL-60) × 45 min, 37°C, 5% CO2 with shaking | This study |
| 2B | % killing of bacteria at indicated serum dilutions | Manual | 1–4 | HBSS + 0.1% gelatin + 5% FBS | 200:1 | (Test serum + target bacteria) × 30 min, 37°C, 5% CO2 with shaking | 10% BRC × 30 min, 37°C, 5% CO2 with shaking; HL-60, × 45 min, 37°C, 5% CO2 with shaking | This study |
The times of incubation with BRC and HL-60 cells are indicated. In OPKA-1B and OPKA-2B, bacteria are incubated with BRC and then incubated with HL-60 cells for the indicated time periods. In assays OPKA-1A and OPKA-1B, exactly the same total amount of BRC was used, but since the aliquot of phagocytes was present when the BRC was added in OPKA-1A, its concentration is less than in OPKA-1B, where the phagocytes were added after the initial incubation with BRC. OPKA-1B and OPKA-1C contain the indicated modifications of OPKA-1A, and OPKA-2B contains the indicated modifications of OPKA-2A.
E/T is the ratio of HL-60 cells to target bacteria.
RT, room temperature.
The concentrations of BRC used in the different assay conditions were tested to ensure functional complement activity at the concentrations used.