A fluorimetric assay for native C3. The hemolytically active form of the third component of human complement

The content of native C3 in samples of purified C3 may be accurately determined using the fluorescent probe ANS (8-anilino-1-napthalene sulfonate). The assay is based on the 11.5-fold increase in fluorescence intensity of ANS which accompanies proteolytic conversion of native C3 to C3b. The assay may be performed in the presence of hemolytically inactive derivatives of C3 such as C3b and C3(H2O). It exhibits the unique feature of being independent of protein concentration and it does not require a C3 standard, other purified complement components, C3 depleted serum, cells or cell-bound intermediate complexes, such as EAC142. A method utilizing cation exchange chromatography (Mono S, Pharmacia) is also described for the rapid (30 min) analytical or preparative separation of native C3 from inactive forms of C3 and from C3 fragments.