Establishment of an enzyme-linked immunosorbent assay (ELISA) for measuring cellular MAGE-4 protein on human cancers.

The MAGE genes encoding tumor-rejection antigens are expressed on various human cancers. An enzyme-linked immunosorbent assay (ELISA) was established for measuring cellular MAGE-4 protein (MAGE-4a and/or -4b) expressed on human tumor cells using a monoclonal antibody (mAb) and polyclonal Ab to recombinant MAGE-4b protein. Both the R5 mAb (IgG1) and the polyclonal Ab recognized a 45 kDa protein in extracts of MAGE-4 mRNA positive cancers, and showed no apparent cross-reactivity to the other MAGE gene products (MAGE-1, -2, -3, -6, and -12) by the immunoblot analyses. The R5 mAb and the polyclonal Ab primarily recognized one (the position 119-133) and two oligopeptides (the positions 119-133 and 259-273), respectively, among a series of 31 different MAGE-4b oligopeptides. The amino acid sequences of these two peptides were identical to those of MAGE-4a and -4b, but differed from those of all the other MAGE proteins (MAGE-1, -2, -3, -6, and -12). Substitution of glycine for amino acid in position 123 (arginine, R), 124 (lysine, K), 126 (R) or 128 (K) in a MAGE-4b oligopeptide of the position 119-132 severely decreased the reactivity of the R5 mAb to the oligopeptide. This ELISA also showed no apparent cross-reactivity with the other MAGE gene products (MAGE-1, -2, -3, -6, and -12). The minimum detectable level of MAGE-4 protein was determined to be 10 pg/well (100 pg/ml). The results suggest that this ELISA is a reliable and quantitative method to measure cellular MAGE-4 protein that is a potential target molecule for specific immunotherapy of human cancers.