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From the Genetics Laboratory, University of Oxford, South Parks Road, Oxford 0X1 3QU, England
Abstract
The experiments reported here show that W6/32 antigenic activity is retained in papain-solubilized HLA antigens and that the W6/32 antibody provides a useful standard reagent to detect and assay HLA-A,B,C antigens. The W6/32 antibody was purified and used to construct an immunoaffinity column. Soluble HLA-A,B,C antigens from papain or detergent-treated membranes could be highly purified in a single step with this column and serologic analysis showed that HLA-A,B and C antigens were bound to the column. Thus, the W6/32 antigenic determinant is present on gene products of all three loci.
The amount of HLA-A,B,C antigens on the surface of human B cell lines and peripheral blood lymphocytes was measured with W6/32 antibody. B cell lines expressed, on average, 9 times as much cell surface HLA-A,B,C antigens as peripheral lymphocytes, although there was appreciable variation within each group. The B cell line Bri 8, for example, expressed 1.5 x 106 W6/32 antigenic sites, and by inference, HLA-A,B,C molecules per cell. Equal amounts of 
2m and HLA-A,B,C chain were found on both cell types.
The isolated HLA-A chain from intact 125I-HLA-A2 antigens weakly bound to W6/32 antibody in contrast to 125I-2-microglobulin (2m) isolated from the same preparation of HLA-A2 antigens that showed no demonstrable binding. when an excess of cold 2m was added to the isolated 125I-HLA-A2 chain, the binding to W6/32 antibody was considerably enhanced. These results suggest that the W6/32 antigenic determinant involves only amino acids of the HLA-A,B,C chain and is a product of their three dimensional configuration. Stable maintenance of this configuration appears to be dependent on the association of the HLA-A,B,C chain with 2m.
Footnotes
1 This work was supported by a grant from the Medical Research Council.
2 Peter Parham was supported by a Junior Fellowship from The Society of Fellows, Harvard University. Please address correspondence to Peter Parham, Sidney Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115.
3 Colin Barnstable was the recipient of a Medical Research Council training award. Present address: Harvard Medical School, Department of Neurobiology, 25 Shattuck Street, Boston, Massachusetts 02115.
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