Complement Components and Their Activation Products in Pleural Fluid: Methods

21 Jun

Complement Components and Their Activation Products in Pleural Fluid: MethodsA diagnostic thoracentesis was done and pleural fluid samples were drawn into tubes containing sodium-EDTA and then centrifuged. The cell-free supernatant was divided into aliquots, frozen, and stored at — 70°C. The complement components (C3, C4, factor B), their activation products (C3d, C4d, Bb), soluble terminal complex (SC5b-9), and soluble Cl/Cl-esterase inhibitor complex (Cls-Clr-CIINH) were analyzed. To determine whether the levels of pleural fluid complement fragments were related to plasma values, plasma specimens were also analyzed canadian pharmacy mall. The plasma samples were taken atraumatically. The first 3 to 4 mL of blood was thrown away and thereafter EDTA-plasma samples were taken. The samples were laid on an icebed and in the course of 1 h, the samples were divided into four portions (0.5 mL). They were then stored at — 70°C.
Complement Complex Determination
C3 and C4 were measured with the turbidimetric system (Turbiquant; Behringwerke; Marburg, Germany) and factor B with the immunodiffusion technique (LC-partigen-C3-Activator; Behringwerke). The activation products of C4 (C4d) and FB (Bb) were measured with the commercial enzyme immunoassay (EIA) method based on monoclonal antibodies (Quidel; San Diego, CA). C3d was measured with rocket electrophoresis ad modum Ramentol-Massana et al. A sandwich enzyme-linked immunosorbent assay for the quantification of soluble SC5b-9 in plasma and pleural fluid was developed according to Gawryl et al. In brief, 96-well flat-bottom plates (Nunc 2-69620 EIA microtiter plate; Nunc, Denmark) were coated with 100 jjlL of goat antihuman C9 (IgG fraction, 30 μg/mL in phosphate-buffered saline solution [PBS]; INC Immunochemicals; Costa Mesa, CA) for 18 h at 37°C. Unbound IgG was removed by washing the plates two times with PBS; thereafter the plates were incubated for 2 h at 37°C with PBS containing 1% bovine serum albumin (BSA) added as blocking agent, and washed twice with PBS. The standards (activated human serum, 8 dilutions 1:3 from 1:10 on) and the samples (1:10) were diluted in 1% BSA-PBS containing 10 mM EDTA to prevent complement activation in vitro; 75 (jlL was pipetted to microtiter wells.