DETECTION OF MEMBRANE-BOUND HLA-G TRANSLATED PRODUCTS WITH A SPECIFICMONOCLONAL-ANTIBODY

A monomorphic anti-HLA-G monoclonal antibody (mAb) was obtained by imm unization of HLA-B27/human beta(2)-microglobulin double-transgenic mic e with transfected murine L cells expressing both HLA-G and human beta (2)-microglobulin, This mAb, designated BFL.1, specifically recognizes , by flow cytometry analysis, the immunizing HLA-G-expressing cells, w hereas it does not bind to parental untransfected or to HLA-B7- and HL A-A3-transfected L cells, suggesting that it distinguishes between cla ssical HLA-A and -B and nonclassical HLA-G class I molecules, This was further assessed by the absence of BFL.1 reactivity with a number of human cell lines known to express classical HLA class I proteins, In a ddition, we showed that the BFL.1 mAb also labels HLA-G-naturally-expr essing JEG-3 and HLA-G-transfected JAR human choriocarcinoma cell line s as well as a subpopulation of first-trimester placental cytotrophobl ast cells. Further biochemical studies mere performed by immunoprecipi tation of biotinylated membrane lysates: BFL.1, like the monomorphic W 6/32 mAb, immunoprecipitated a 39-kDa protein in HLA-G-expressing cell lines, a size corresponding to the predicted full-length HLA-G1 isofo rm, However, in contrast to W6/32, which immunoprecipitates both class ical and nonclassical HLA class I heavy chains, BFL.1 mAb does not rec ognize the class Ia products, Such a mAb should he a useful tool for a nalysis of HLA-G protein expression in various normal and pathological human tissues and for determination of the function(s) of translated HLA-G products.