Human C4 is most polymorphic at the protein level, distinction between allotypes of the C4A and C4B proteins resting on electrophoretic migration patterns and difference in hemolytic activity. The aim of the C4 reference typing has been the definition of reference variants, the assignment of rare variants, and the investigation of duplicated, deleted, or non-expressed and hybrid genes. Samples from 136 individuals, predominantly with known segregation, from 16 laboratories were investigated by standard electrophoretic techniques, for their relative hemolytic activity, reactivity with monoclonal antibodies and Rg/Ch reagents, a-, and ß-chain types, relative electrophoretic migration distance, as well as the C4/21-OH-TaqI RFLPs. The results were evaluated in three groups; they consisted in the definition of the eight most common C4 alleles, and the ten Rg/Ch standard phenotypes in group I. In group II twelve C4A and fourteen C4B duplications among 96 complotypes, as well as eighteen deleted/non-expressed C4A and twenty-two C4B alleles, and hybrid alleles were seen by correlation of lytic activity, electrophoretic mobility, and monoclonal and/or Rg/Ch reactivity. Group III consisted of the newly defined allotypes A 8, A 7, A 58, A 55, A 45, B 45, B 35, and B 22, furthermore of alleles subdividing the A 1/A 91, and the B 13/ B 12/B 11 regions. The reference typing has allowed reclassification of the majority of described C4 allotypes and resulted in a revision of the C4 nomenclature.

In a factor B (BF) Reference Typing of the Vlth Complement Genetics Workshop and Conference, Mainz, FRG, 1989, 99 samples from 13 laboratories, including 18 families, were investigated with the majority of presently known typing procedures. Among the major (‘standard’) allotypes BF S04 was found to be new. For the group of common BF F subtypes samples from 11 laboratories including complete family data from 5 laboratories were compared. The subtypes BF FA and FB were recognized and confirmed to be identical in the samples from all groups. Within a third group rare subtype variants of F and S were compared and characterized. In samples submitted from individuals with assumed non- expressed (BF*Q0) alleles unexpected and hypomorphic gene products were seen. The investigation of DNA samples for restriction fragment length polymorphisms from the same set of individuals revealed a correlation of the Msp 10.7-kb fragment with BF F, and confirmed the correlation of a Taq I 6.6-kb fragment with BF FA.

For the definition of common C4 allotypes relative electrophoretic migration (RM) values were determined. A set of standard C4 variants were investigated by prolonged agarose gel electrophoresis and subsequent immunofixation with specific antiserum. RM distances were measured by laser densitometry. Using an arbitrary standard of 100 units for the migration distance between the C4B 1 and C4A 3 bands a total deviation of ± 6.45% in more than 108 single determinations was found. The common C4 alleles used for standardization were C4A*6, C4A*4, C4A*3, C4A*2, C4B*5, C4B*3, C4B*2, C4B*1. In addition, side by side comparison and admixture of known variants will be necessary for the differentiation of some of the closely migrating allotypes. RM values are now available for eight alleles frequently found in all populations for future comparison and designation of newly discovered C4 allotypes.