11] Preparation of human factor [formula omitted] of the alternative pathway of complement

Abstract

Publisher Summary This chapter presents the procedure for purification and assaying of human Factor D. Outdated human plasma can be used to prepare satisfactory yields of Factor D but it is preferable to use fresh or flesh-frozen plasma because material having a similar molecular weight and similar ion-exchange properties may be generated on prolonged storage of plasma or even of the partially purified fractions during the purification procedure. Addition of protease inhibitors in the early stages of the procedure did not appear to increase the recovery of Factor D. Plasma or serum may be used in the method in this chapter, the yield and final specific hemolytic activity of factor D being the same in both cases. When serum was used it was prepared from the plasma by clotting overnight at 4°C on the addition of 1.0 M CaC1 2 to give a final concentration of 20 mM. The clot was removed by centrifugation and filtration through muslin. The hemolytic activity of factor D involves the alternative pathway dependent lysis of rabbit erythrocytes in solution, or by alternative pathway-dependent lysis of guinea pig erythrocytes on an agarose plate. The factor D hemolytic activity is determined by the extent of hemolysis at 412 nm.