Restricted rosette formation with an EAC reagent prepared with serum of C3H/HeJ mice

Estimation of classical pathway of mouse complement activity by use of sensitized rabbit erythrocytes

“…In one experiment mouse plasma was used obtained by mixing 40 pl of a millipore-sterilized Mg-EGTA stock solution, pH 7.4 (containing 0.5 M MgSO4 and 0.2 M ethyleneglycol-bis-(2-aminoethyl)-tetraacetic acid; Van Dijk et al, 1980a), with 0.96 ml fresh mouse blood and subsequent centrifugation. For recalcification 200 pl plasma were diluted 1 : 10 in the test buffer with an admixture of 0.8 mM calcium.…”

“…In the original assay, analogous to that for mouse alternative C pathway determination (Van Dijk et al, 1980a), 200 pl of mouse (or guinea pig) serum serially diluted in VSB 2÷ were mixed and subsequently incubated for 30 min at 37°C with 100 pl of RaRBC-A, MoRBC-A or ShRBC-A (1.5 × l0 s cells/ml). To stop the reaction 2.2 ml of ice-cold VSB 2÷ were added, after which the cells were spun down and the E412 of the supernatant was measured.…”

“…To determine the optimal conditions for the assay of mouse classical C pathway activity the incubation temperature, time and volume of the original method according to Takada et al (1978) and Van Dijk et al (1980a) were varied and moreover the cell numbers per test tube. Incubation temperatures of 22--30°C {Fig.…”

“…Although modification to 51Cr-release tests greatly improved the results for mouse serum (Berden et al, 1978), this adaptation did not eliminate the great difference in efficiencies between mouse and, e.g., guinea pig C. However, with rabbit erythrocytes (RaRBC) as target cells no great differences between alternative C pathway activities of mouse and guinea pig serum were measured (Van Dijk et al, 1980a). As alternative C pathway activation by RaRBC is thought to occur by hindrance of the C3b inactivator system (Fearon and Austen, 1977), this system is most probably responsible for the failing lysis of ShRBC by mouse serum, which was already suggested by Borsos and Cooper in 1961. The relative unsensitiveness to the C3b inactivator system prompted us to study the classical C pathway activity of mouse serum using sensitized RaRBC (RaRBC-A) as target cells.…”

“…As alternative C pathway activation by RaRBC is thought to occur by hindrance of the C3b inactivator system (Fearon and Austen, 1977), this system is most probably responsible for the failing lysis of ShRBC by mouse serum, which was already suggested by Borsos and Cooper in 1961. The relative unsensitiveness to the C3b inactivator system prompted us to study the classical C pathway activity of mouse serum using sensitized RaRBC (RaRBC-A) as target cells. CH50 values thus obtained were compared to similar values from tests using sensitized mouse erythrocytes (MoRBC-A), which are activators of the human alternative C pathway (Kazatchkine et al, 1979;Van Dijk et al, 1980a) and sensitized ShRBC (ShRBC-A). Classical pathway of mouse C activation by RaRBC-A was studied in more depth.…”