The detection of HLA-DR, MB and MT determinants on purified class II molecules by inhibition of microcytotoxicity.

An assay has been developed which makes it possible to determine the HLA allospecificities carried by molecules in purified fractions of detergent lysates from EBV-transformed human lymphocytes. It is based on inhibition of the standard microlymphocytotoxic test used for identifying HLA class I and II antigens with alloantisera. Soluble cell membrane products from EBV-transformed cell lines homozygous for the HLA region gave specific inhibition of standard typing antisera. The test requires preincubation of microliter volumes of soluble antigen preparations maintained in 0.05% NP-40 with selected antisera prior to adding EBV-transformed cells as target cells. It was possible using this assay to follow isolation of the structurally related human class II molecules bearing the MB and DR specificities. Detergent lysates of cells were fractionated on affinity columns prepared from monoclonal antibodies directed against distinct class II antigens. Eluates from these columns contained the expected DR and MB specificities. The assay is easy to perform, highly reproducible and allows multiple determinations.