Purification and use of the C3d subunit C3.
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 hydroxy-apatite. The highly purified C3 and C3b so obtained are then treated with commercial C3b inactivator. G-200 sephadex filtration of this material produces a low molecular weight peak containing C3d greater than 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 less than 70 mug 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 C5b. Two of three rabbits hyperimmunized 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 glutinated 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 IgG. Preliminary studies with 125I-labeled C3d indicate its potential value in assessing potency of anti-C3d reagents.
D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003176:Complement C3 / Q000302:isolation & purification*; D003165:Complement System Proteins / Q000302:isolation & purification*; D004912:Erythrocytes / Q000276:immunology; D006801:Humans; D007114:Immunization; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D011234:Precipitins / Q000096:biosynthesis; D011817:Rabbits
D000888:Antibodies, Anti-Idiotypic; D003176:Complement C3; D007457:Iodine Radioisotopes; D011234:Precipitins; D003165:Complement System Proteins