Development and characterization of rabbit antisera to human MHC-linked transporters associated with antigen...

The limited availability of sera to human MHC-linked transporters associated with antigen processing (TAP) has hampered the analysis of the role of these molecules in the reduced HLA Class I antigen expression by normal and transformed cells. To overcome these limitations, anti-human TAP1 and anti-human TAP2 xenoantisera have been generated and characterized. To this end rabbits have been immunized with TAP1-specific or TAP2-specific peptides which correspond to nonhomologous, hydrophilic regions of each transporter subunit. The immunized rabbits developed high titer IgG antibodies which displayed specific reactivity with the immunizing peptides in ELISA. Both anti-TAP1 and anti-TAP2 antisera immunoprecipitated the 70-76 kDa TAP complex from TAP(+)-TAP2+ cell lines WALK and Colo 38, but precipitated no component from TAP(-)-TAP2- cell lines T2 and SK-MEL-19. Furthermore, in immunodepletion experiments anti-TAP1 and anti-TAP2 antisera removed the molecules recognized by each of them in a lymphoid cell extract. Lastly, in Western blotting assays anti-TAP1 and anti-TAP2 antisera reacted specifically with isolated TAP1 and TAP2, respectively. The latter results in conjunction with those of the immunodepletion experiments indicate that TAP1 and TAP2 are not detectable as isolated subunits in a cell extract and that TAP heterocomplex is the major, if not the only detectable molecular species in cells. Anti-TAP1 and anti-TAP2 antisera were evaluated in immunohistochemical staining of both frozen and formalin fixed sections of skin and primary malignant melanoma lesions. Both antisera stained the cytoplasm of keratinocytes in normal skin and of melanoma cells in malignant lesions. The antisera we have elicited with TAP1- and TAP2-specific peptides appear to be useful reagents to characterize the role of TAP in abnormalities of HLA Class I antigen expression.