A combined approach for quantitation of each specific HLA-A or -B antigen expressed on cells

In order to measure the amount of each individual HLA-A or -B antigen expressed in or on a cell without relying on monoclonal antibodies to different specific HLA antigens, we have developed a combined approach that consists of two separate measurements. The first measurement is to determine the relative quantities of different HLA-A and -B antigens in lysates of whole cells using IEF gel electrophoresis, immunoblotting with 171.4 anti-HLA heavy chain monoclonal antibody, and scanning densitometry. The second measurement is to determine the concentration of total HLA antigens expressed in or on a cell using an enzyme-linked immunoassay or a surface binding assay based on W6/32 anti-HLA monoclonal antibody. The quantity of each specific HLA antigen in or on a cell then is calculated from the results of these two measurements. To validate this combined approach, we conducted studies to show that the amounts of different specific HLA antigens measured by this approach were linearly correlated with those measured by FITC-labeled monoclonal antibodies and immunofluorescence flow cytometry in platelets and lymphoblastoid cell lines. We also demonstrated that the relative quantities of different HLA-A and -B antigens determined from the lysates of whole cells were the same as those expressed on the cell surface. These findings indicated that the newly developed combined approach can be applied to quantify each specific HLA-A or -B antigen expressed in or on a cell.