Immunoblot analysis of the eighth component of human complement. Demonstration of subunits and detection of C8 alpha-gamma double and triple bands.
Using SDS-PAGE/immunoblot analysis of the eighth component of human complement, C8, we have been able to demonstrate an 85 kDa C8 alpha-gamma and a 62 kDa C8 beta 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 C8 beta subunit. Serum of a patient with known C8 alpha-gamma 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 C8 alpha-gamma subunit was found to be composed of up to three bands, termed C8 alpha-gamma 1, -2 and -3. All three bands were found in pooled normal sera. Individual sera had at least the C8 alpha-gamma 2 and C8 alpha-gamma 3 bands. Two C8 beta-deficient sera from two unrelated patients exhibited only the C8 alpha-gamma 2 and C8 alpha-gamma 3 bands. We conclude that immunoblotting of C8 permits a detailed analysis of the molecular composition of this component and helps to establish a precise diagnosis in inherited C8 deficiencies.
Nürnberger
W
W
Department of Neonatology and Gastroenterology, University of Düsseldorf, F.R.G.
Seger
R
R
Kobler
P
P
Mons
I
I
Tedesco
F
F
Wahn
V
V
D003101:Collodion; D003185:Complement C8 / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010209:Paper; D011487:Protein Conformation
D003185:Complement C8; D003101:Collodion