The use of conglutinin in a quantitative assay for the presence of cell-bound C3bi and evidence that a single molecule of C3bi is capable of binding conglutinin.
We have developed a quantitative assay for cell surface C3bi using 125I-labeled conglutinin. Conglutinin was purified to homogeneity from bovine serum and radiolabeled with 125I Bolton Hunter reagent. Conditions of time, temperature, ionic strength, and cell concentration that optimized the binding of conglutinin to erythrocytes bearing C3bi were then determined. The interaction between conglutinin and C3bi under these conditions was highly specific, since EAC4b3b, EAC4b3d, EAC4b3b-beta IH, and EAC4b treated with serum did not bind radioconglutinin significance better with EA or EAC4b. Using this assay, was examined the kinetics of inactivation of both human and guinea pig C3b bound to erythrocytes and showed that, for both, maximum conglutinin binding occurred after EAC4b3b had been incubated with a source of beta 1H and C3INA for 10 to 20 min at 37 degrees C.l We showed a linear relationship between the number of molecules of C3bi per erythrocyte and the amount of conglutinin bound for both guinea pig and human C3bi. The affinity of conglutinin for cell-bound C3bi was shown to be independent of C3bi density on the erythrocyte surface, and the Kd for conglutinin binding to erythrocytes bearing human C3bi was determined to be 1.3 X 10(-8) M. The number of conglutinin binding sites per erythrocyte as calculated from Scatchard plots was equal to the number of C3bi molecules on the cell surface as determined by direct assay using 125I-labeled C3. Moreover, for both human and guinea pig C3bi, the plot of log (cell surface C3bi) vs log (conglutinin bound) had a slope of 1. These findings imply that a single molecule of C3bi is capable of binding a molecule of conglutinin under the conditions of our assay.
Brown, EJ; Gaither, TA; Hammer, CH; Hosea, SW; Frank, MM
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