A METHOD FOR ASSESSING THE REASSEMBLY OF A MULTISUBUNIT GLYCOPROTEIN BY WESTERN BLOTTING

The Western blot procedure has been adapted to detect the reassembly of a two-subunit glycoprotein, urinary human chorionic gonadotropin (hCG), directly on the nitrocellulose. This glycoprotein is composed of two nonidentical subunits, α and β. A simple procedure using immunoblotting has been developed to detect reassembly of the monomers to dimer. Three monoclonal antibodies were required for the development of this method: A109, which binds the α subunit or hCG; B105, which binds the β subunit or hCG; and B107, specific for the intact hCG dimer. The α subunit and β subunit of hCG were each electrophoresed and transferred to nitrocellulose, and the transfer was then incubated with the appropriate complementary subunit; reassembly of the dimer was determined by the binding of the monoclonal antibody B107. Evidence that the reassembly occurs directly on the nitrocellulose comes from the fact that B107 immunoreactivity is detected at the molecular weight position of the subunit and not at the dimer molecular weight. A genetically expressed recombinant form of the α subunit was also tested for its ability to recombine with the opposite subunit to produce the dimer. The recombinant α subunit was determined to have additional carbohydrate which interfered with the binding of the β subunit. N-Glycanase digestion of the recombinant α subunit produced a form which, when incubated with the β subunit, did recombine on the nitrocellulose and could be recognized by B107. © 1991.