Immunoblot analysis of the eighth component of human complement: Demonstration of subunits and detection of C8α-γ double and triple bands

Abstract

Using SDS-PAGE/immunoblot analysis of the eighth component of human complement, C8, we have been able to demonstrate an 85 kDa C8α-γ and a 62 kDa C8β subunit in normal human serum. Serum from an undiagnosed patient who presented undetectable hemolytic C8 activity possessed only the 85 kDa subunit, suggesting a defect in the C8β subunit. Serum of a patient with known C8α-γ deficiency possessed only the complementary 62 kDa subunit. Both sera used together were able to lyse antibody-sensitized sheep erythrocytes, whereas individual sera could not. Optimum conditions for C8 immunoblotting were determined using small amounts of serum or plasma, during low voltage electrophoresis and a sensitive staining technique (nitrobluetetrazolium/bromochloroindoxylphosphate). Using these conditions, the C8α-γ subunit was found to be composed of up to three bands, termed C8α-γ1, -2 and -3. All three bands were found in pooled normal sera. Individual sera had at least the C8α-γ2 and C8α-γ3 bands. Two C8β-deficient sera from two unrelated patients exhibited only the C8α-γ2 and C8α-γ3 bands. We conclude that immunoblotting of C8 permits a detailed analysis of the molecular composition of this component and helps to establih a precise diagnosis in inherited C8 deficiencies.