CHARACTERIZATION OF THE 2 UNIQUE HUMAN ANTI-FLAVIN MONOCLONAL IMMUNOGLOBULINS

Form A of two previously described human monoclonal anti-riboflavin Ig Gs, the GAR [Farhangi, M. and Osserman, E. F. (1976) N. Engl. J. Med. 294, 177-183] and DOT [Merlini, G., Bruening, R., Kyle, R. and Osserma n, E. F. (1990) Mol. Immunol. 27, 385-394], has been characterized in terms of binding properties and primary structure. Both forms were iso lated as immunocomplexes with bound riboflavin and gave a reconstituta ble apoprotein. The riboflavin-reconstituted IgGs showed a similar vis ible absorption spectrum, with a marked resolution of the 445-nm band and a ratio 445-nm/370-nm peaks of 1.13 for DOT and 1.19 for GAR. Both proteins bind riboflavin, FMN and FAD with a molar ratio ligand/prote in of 2:1. DOT and GAR share a very similar affinity for the flavinic ligands; the K-d values for riboflavin and FMN are in the range 1 nM; that for FAD is an order of magnitude higher. DOT and GAR do not form an adduct between the nucleophilic group sulfite and the N(5) position of the flavin, and do not stabilize any flavinic semiquinone during r eduction with the xantine/xantine oxidase benzylviologen system. The p rimary structure of fragment antigen binding (Fab) DOT and heavy-chain variable region (V-H) GAR determined in the present study and that al ready known for the light-chain variable region (V-L) GAR [Kiefer, C. R., McGuire, B. S., Osserman, E. F. and Garver, F. A. (1983) J. Immuno l. 131, 1871-1875] evidenced that the two IgGs are assembled with V-L and V-H chains of different subgroups; a lambda III/HIII pair in GAR, and a lambda II/HI pair in DOT. Although less similar each other than to the counterparts of the same subclasses, DOT and GAR share an exclu sive identity in the V-H CDR3 region.