HLA class I biochemistry: definition and frequency determination of subtypes by one-dimensional isoelectric focusing and immunoblotting.

Previous biochemical studies of HLA class I gene products were based on the use of radiolabelled lymphoblastoid cell lines and/or mitogen-stimulated peripheral blood lymphocytes (PBL). We present here a nonradioactive methodology for defining HLA class I polymorphism on a small number of cells, such as 5 x 10(6) lymphocytes, the vitality of which is not a requirement in contrast to radiolabelling techniques, where 1-2 x 10(6) PBL are needed. We analyzed 54 serologically typed families with a total of 261 individuals and 214 haplotypes by one-dimensional isoelectric focusing and subsequent immunoblotting, using a polyclonal anti-class I heavy chain serum. The banding patterns segregated within families and correlated with the serological results. A substantial number of HLA-A and HLA-B products appeared biochemically heterogeneous allowing the definition of subtypes and the calculation of their frequencies.