METAL-BINDING PROPERTIES OF A CALCIUM-DEPENDENT MONOCLONAL-ANTIBODY

The calcium-dependent mAb, M1 (also called anti-Flag or 4E11) was stud ied using a newly developed metal-sensitive enzyme-linked immunosorben t assay (ELISA). This antibody, specific for a calcium complex of the peptide antigen, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, has found widespread use as a mild purification reagent for Flag-epitope tagged recombinan t proteins. Although M1 affinity columns release monovalent Flagged pr oteins in the absence of calcium, the antibody retains substantial aff inity for the Flag sequence even in metal-free conditions, so that it has been impossible to use it to develop a metal-sensitive ELISA assay . This is due to the ability of the antibody to remain bound to polyva lent surface-coated antigen, for instance, when Flagged proteins are b ound to ELISA plates or blotting filters. The resultant antigen polyva lence raises the avidity of the Flag antibody to a point where the rea ction is essentially calcium-independent. However, when the antibody i tself was made monovalent, by proteolytic cleavage to the Fab, this si tuation was reversed and the ELISA reaction became calcium-dependent, This new metal-dependent ELISA assay was used to explore the metal req uirements of the antibody in detail. Among divalent metals, binding ta pered off with increasing radius above that of calcium, or with decrea sing radius below that of calcium. Several smaller metals, such as nic kel, acted as inhibitors of the binding reaction. Substantial binding was demonstrated for heavy metals such as cadmium, lanthanum and samar ium. Because it is of interest to use this antibody for the co-crystal lization of recombinant Flag-fusion proteins, the ability to bind heav y metals was a significant finding. Copyright (C) 1996 Elsevier Scienc e Ltd.