Complement Components and Their Activation Products in Pleural Fluid: Complement Complex Determination

23 Jun
2014

After 2 h of incubation at 37°C, and washing three times with distilled water containing 0.1% polysorbate (Tween) 20 (aqua-Tween), rabbit-antihuman C5 (Behringwerke) diluted 1:200 in 1% BSA-PBS was added for overnight incubation at room temperature. After washing three times with aqua-Tween, antirabbit-IgG antiserum (Orion Diagnostics Espoo, Finland) labeled with alkaline phosphatase diluted 1:300 with 1% BSA-PBS was added for 4 h incubation at 37°C. After washings, 100 \xh of 1,2-p-nitrophenyl phosphate, 1 mg/mL (Reagena; Kuopio, Finland), in diethanol-amine buffer (pH 10.5) was added and the reaction was stopped after 30 min with 1 M NaOH. The color was read (with Multiskan; Labsystems; Helsinki, Finland) at 405 nm and the results were given as AU/mL (percent of the standard). Cl activation was assessed by quantification of soluble Cls-Clr-C1INH by enzyme-linked immunosorbent assay. In brief, 96-well flat-bottom plates (Nunc 2-69620 EIA microtiter plate; Nunc, Denmark) were coated with 100 μL of goat antihuman Cls (IgG fraction, 40 μg/mL; Atlantic antibodies) for 18 h at 37°C, and then washed twice with PBS. To prevent nonspecific binding, blocking was done with 1% BSA-PBS for 2 h at 37°C. Buy birth control More info To prevent complement activation during determination, the samples were diluted 1:5 in 1% BSA-PBS containing 0.01 M EDTA. After 2 h of incubation at 37°C, washing was done three times with distilled water containing 0.1% polysorbate 20. Thereafter, 100 (μL of rabbit antihuman Cl inactivator (Behringwerke) diluted 1:400 in 1% BSA-PBS was added for overnight incubation at room temperature. After washing three times with Aqua-tween, antirabbit-IgG antiserum labeled with alkaline phosphatase (Orion Diagnostica) diluted 1:300 with 1% BSA-PBS was added for 4 h of incubation at 37°C. After washings, 100 μL of 1,2-p-nitrophenyl phosphate (Reagena) 1 mg/mL in diethanol-amine buffer (pH 10.5) was added and the reaction was stopped after 30 min with I M NaOH. The color was read (with Multiskan; Labsystems) at 405 nm and the results were given as AU/mL (percent of the standard). The standard preparation for the soluble Cls-Clr-CIINH complexes was prepared by incubating normal human serum pool with heat-aggregated human IgG (2 mg/mL) for 60 min at 37°C. After activation, 0.01M EDTA was added to prevent further activation.

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