Serological typing of HLA-C antigens is rather difficult mainly because HLA-Cw blank antigens are present at the gene frequency of 30-50% in most human races. Here we have estabished HLA-C middle-high resolution DNA typing as a simple and sensitive technique, using the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method, which allows to classify HLA-C alleles into 18 groups by 13 restriction enzymes. HLA-C alleles defined by PCR-RFLP developed here in 26 HLA-homozyous cell lines were in complete agreement with those previously assigned by serological and or DNA typing except one cell, revealing that this PCR-RFLP method is useful for analysis of HLA-C alleles in HLA-disease association studies and HLA matching for donor selection in unrelated bone marrow transplantation.

To analyze HLA-B antigens by isoelectricfocusing, multiple antigenic peptide (MAP) of the 16 amino acid sequece specific for HLA-Bα1 domain was prepared and immunized. The reactivity of the anti-HLA-Bα1 MAP serum with HLA-A, -B antigens was examined by isoelectricfocusing-immuno blot (IEF-IB). Of HLA-B antigens, the anti-serum strongly reacted with HLA-B 13, -B44, -B48. -B52, -B60, -B61, -B62 which had the identical amino acid sequence with the immunogen. But it did not react with HLA-B7, -B8, -B14, -B18, -B27, -B35, -B46, -B51, -B53, -B54, -B55, -B70, -B75 whose amino acid difference from the immunogen was in the range of 1〜6 out of the 16 amino acids. Of HLA-A antigens, it did not react with either of the HLA-A antigens examined, except that is weakly reacted with HLA-A 2, -A24. Immunization of synthetic MAP was useful to prepare anti-serum specific for HLA-B antigens for IEF-IB. Also, the anti-serum was considered to be an excellent reagent for biochemical analysis of HLA-B antigens.