Mild acid treatment induces cross-reactivity of 4H84 monoclonal antibody specific to nonclassical HLA-G antigen with classical HLA class I molecules

Abstract

Mild acid treatment by releasing β 2m and antigenic peptides leaves human leukocyte antigen (HLA) class I free heavy chains attached to the cell surface. Acid treatment thus allows detection of the cell surface class I antigens by monoclonal antibodies (mAbs) specific to HLA-free heavy chains. We found that acid treatment also enables detection of the cell surface non-classical HLA-G class I antigen with mAbs specific for HLA-G free heavy chains, including 4H84 mAb recognizing all isoforms. Furthermore, we found that 4H84 mAb, but not other mAbs specific to HLA-G free heavy chains, binds to the surface of 8 out of 16 acid-treated leukemia cell lines. Nevertheless, HLA-G antigen is not present in any of these leukemia cells. This was demonstrated by failure to detect any antigen with 4H84 mAb in immunoblotting as well as by inability to detect HLA-G mRNA by RT-PCR. The antigen recognized by 4H84 mAb in some acid treated leukemia cells was identified by immunoprecipitation as a 45 kDa protein. A number of observations indicate that 45 kDa proteins are none other than classical class I heavy chains. Acid treatment thus induces the ability of the 4H84 mAb to recognize some classical HLA class I molecules. Remarkably, 4H84 determinant on HLA-G is linear but corresponding determinant present on some partially folded classical HLA class I free heavy chains is conformational. In view of the unexpected cross-reactivity, detection of HLA-G with this mAb must be carefully evaluated to avoid false detection.