S1389

Normal human serum (NHS) used as a source of complement was collected by medical staff (Laboratory of Viral Infections Diagnostics, Department of Clinical Microbiology, Pavlov First Saint Petersburg State Medical University, Saint Petersburg, Russia) from more than 20 healthy volunteers, pooled, aliquoted, and stored at −70 °C no longer than two months. Serum aliquots were thawed at +4 °C on the day of the experiment, kept in an ice bath before introducing to test tubes, and were not used repetitively. To obtain serum with inactivated complement, it was incubated at +56 °C for an hour immediately before the experiment.
Animal erythrocytes were purified from whole blood of rabbit and sheep and stored in Alsever’s solution at +4 °C. Before use, they were washed with appropriate buffer: DGVB⁺⁺ (dextrose gelatin veronal buffer with Ca²⁺ and Mg²⁺) for sheep erythrocytes (E^sh) and GVB⁺ (gelatin veronal buffer with Mg²⁺) for rabbit erythrocytes (E^rab). DGVB⁺⁺ is 4.5 mM sodium barbital buffer containing 150 mM NaCl, 15 mM glucose, 0.15 mM CaCl₂, 1 mM MgCl₂, 0.05% gelatin; pH 7.4. GVB⁺ is 4.5 mM sodium barbital buffer containing 150 mM NaCl, 10 mM Mg-EGTA, 0.05% gelatin; pH 7.4. Before the experiments, E^sh was sensitized with antibodies (anti-sheep red blood cell stroma antibodies produced in rabbits, S1389, Sigma, St. Louis, MO, USA) diluted 1:1600 for 40 min at +37 °C.

GVB⁺⁺ or Mg EGTA buffers, which had been supplemented with 2.5% glucose (w/v), were used for the CP and AP assays, respectively. For the AP, 150 µL of rabbit erythrocytes (TCS Biosciences, Botolph Claydon, UK) were washed twice, by addition of 1 mL of buffer and centrifugation at 800 ×g for 1 min, and finally resuspended in 500 µL of buffer. For the CP, 150 µL sheep erythrocytes (TCS Biosciences, Botolph Claydon, UK) were washed twice with 1 mL of buffer and sensitised with a 1/1000 dilution of rabbit anti-sheep red blood cell stroma antibody (S1389, Sigma Aldrich, Gillingham, UK). After a 30°C/30 min incubation, with shaking, the cells were rewashed and resuspended with 500 µL of buffer. Serial dilutions of peptide were prepared in the respective buffers and normal human serum was added at 1% for the CP and 4.5% for the AP (corresponding to CH50 of the serum). Also, 90 µL of peptide–serum mixtures were plated into a V-bottom 96-well microtiter plate (Corning) and 10 µL of erythrocytes were added. Plates were incubated for 30 min at 37°C, with shaking. Finally, 50 µL of buffer was added, the plates centrifuged at 800 ×g, and 80 µL of supernatant was transferred to an ELISA plate (Nunc) and absorbance measured at 405 nm.

Normal human serum used as a source of complement was collected from 30 healthy volunteers, pooled, aliquoted and stored at −70 °C. Erythrocytes were purified from whole blood of rabbit, sheep and healthy donors. The fresh blood was mixed with Alsever’s solution (1:2) and stored at 4 °C for no more than 5 days. Before use, we obtained erythrocytes from the blood and washed them with an appropriate buffer: DGVB⁺⁺ (dextrose gelatin veronal buffer with Ca²⁺ and Mg²⁺: 5 mM sodium barbital buffer containing 150 mM NaCl, 15 mM glucose, 1 mM MgCl₂, 0.15 mM CaCl₂, 0.05% gelatin; pH 7.35) for sheep erythrocytes (E^sh); GVB⁺ (gelatin veronal buffer with Mg²⁺: 5 mM sodium barbital buffer containing 150 mM NaCl, 10 mM Mg-EGTA, 0.05% gelatin; pH 7.35) for rabbit erythrocytes (E^rab) and PBS (phosphate buffered saline; pH 7.4) for human erythrocytes (E^hum). Sheep erythrocytes were sensitized with antibodies (anti-sheep red blood cell stroma antibodies produced in rabbits, S1389, Sigma, St. Louis, MO, USA) before use in experiments; we used a 1:1600 dilution of these antibodies and incubated sheep erythrocytes for 30 min at 37 °C.