Purification and Use of the C3d Subunit of C3

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Abstract
A method is described for the preparation of C3d from fresh‐frozen CPD plasma. A redissolved EDTA euglobulin precipitate formed from defibrinated plasma is chromatographed on DEAE cellulose. The C3‐rich peak is further chromatographed by stepwise elution from hydroxyl‐apatite. The highly purified C3 and C3b so obtained are then treated with commercial C3b inactivator. C‐200 sephadex filtration of this material produces a low molecular weight peak containing C3d in > 95 per cent purity. The purified C3d, which contains negligible carbohydrate, has a molecular weight of 28,000 (based on SDS polyacrylamide gel electrophoresis). When added at a final concentration of < 70 μg per ml, it completely inhibits the anti‐C3d hemagglutinin reactivity of a potent anti‐C3d antiglobulin serum but does not inhibit sera with reactivities against C3c, C4b, and CSb. Two of three rabbits hyper immunized with purified C3d produced antisera exhibiting only anti‐C3d precipitin activity. After absorption of heterologous antibodies, the antisera strongly agglutinated C3d‐coated RBC, failed to agglutinate RBC coated only with C4, and reacted weakly against strongly anti‐Rh‐coated RBC. The latter reactivity was readily absorbed by whole IgC. Preliminary studies with 125I‐Iabeled C3d indicate its potential value in assessing potency of anti‐C3d reagents.