Search

Biochemical analysis of HLA-B27 antigens from individuals that are HLA-ABC identical by serology but distinct in CML typing establishes that two types of HLA-B27 can be defined biochemically. The division of HLA-B27 into a W type and a K type by CML typing correlates perfectly with the biochemical data. HLA-B27 K type possesses a more basic isoelectric point than HLA-B27 W type. Neuraminidase digestions of immunoprecipitated HLA-A,B antigens establish that the difference in isoelectric point between HLA-B27 K and HLA-B27 W is not due to differences in sialic acid content.

The population of HLA-A2-positive individuals, currently considered serologically homogeneous, can be divided into three subtypes on the basis of antigen recognition by various HLA-A2-specific cytotoxic T lymphocytes (CTLs). When these three types of HLA-A2 antigens were analyzed biochemically, they were found to be distinct. Isoelectric focusing (IEF) of HLA antigens digested with neuraminidase (NANAse) suggested that the difference(s) reside in the polypeptide backbone of the HLA-A2 heavy chain. Biochemical analysis distinguishes three distinct categories of HLA-A2 antigens: (1) a major subtype, designated HLA-A2.I, (2) a minor subtype, designated HLA-A2.II, possessing a more basic isoelectric point (IEP) and (3) a minor HLA-A2 subtype more acidic in its IEP than HLA-A2.I, designated HLA-A2.III. A fourth HLA-A2 subtype could be defined by discordance between cell-mediated lympholysis (CML) typing and biochemical analysis. The latter HLA-A2 antigen was defined as a variant by CTL, but was biochemically indistinguishable from the major subtype HLA-A2.I.

Class I antigens were isolated by immunoprecipitation from cell extracts prepared from mitogenically stimulated and internally radiolabeled peripheral blood lymphocytes (PLBs). The precipitating antibodies used are monomorphic and recognize a determinant on the heavy chain of HLA-A, B, C antigens regardless of their allelic specificities when complexed with beta 2m, or determinants on beta 2m itself. Comparison of class I molecules isolated from 25 different homozygous typing cells (HTC) and analyzed by two-dimensional (2-D) gel electrophoresis allowed the identification of those HLA-A, B locus specificities most common in the European Caucasoid population. Class I antigens isolated from HTC that are HLA identical are biochemically indistinguishable also. Evidence was obtained for the expression of additional class I antigens besides the HLA-A, B, C locus products: for some haplotypes, up to six class I genes may be active in mitogenically activated PBLs.No differences in molecular weight and isoelectric point of the class I heavy chains were observed between the antigens recognized by W6/32, the anti-heavy chain reagent, and anti beta 2m reagents. The nature of the mitogenic stimulus, i.e., pokeweed mitogen or phytohemagglutinin, was irrelevant with respect to the class I antigens isolated by this method. Using the HTCs as reference, a panel of HLA-B27 positive heterozygous cells was analyzed. Two types of HLA-B27 antigens, distinct by CML typing were represented. These two forms differed also in their biochemical properties. In addition, we obtained evidence for the existence of an A2 variant. This finding was likewise confirmed by CML typing.