Polymorphism of human C2 detected by immunoblotting

To allotype human complement component C2, thin layer agarose gel isoelectric focusing of human serum and/or EDTA-plasma was performed followed by direct immunofixation or by immunoblotting with a specific anti-human-C2 antibody. Using reference samples for C2 BC phenotypes and local samples from an HLA, C4, and Bf genotyped family, a differentiation of the C2*B and C2*C variants segregating with the respective HLA haplotype was achieved. The C2 BC phenotype is characterized by a double banding pattern similar to that observed in the haemolytic overlay assay usually used for the detection of C2 polymorphism. An homozygous C2*Q0 reference sample determined by functional assays was shown to be biochemically deficient, as demonstrated by immunofixation and immunoblotting. The visual interpretation of C2 phenotypes was definitely easier after immunofixation and immunoblotting than after an haemolytic overlay assay. In addition, the method for C2 allotyping described here has several advantages, in particular it saves time and tolerates repeated thawing and freezing of the samples.