In order to explore pathologies possibly associated with vitamin K deficiency, several monoclonal antibodies (mAbs) were produced against human Desgamma-Carboxy-Prothrombin (DCP). One of these mAbs, designated C4B6, detected DCP forms in the presence of Calcium ions, confirmed by comparison with the patterns of two electrophoretic techniques: Affino-Immuno-Electrophoresis (CAIE) and Polyacrylamide Gel Electrophoresis followed by Electro-blotting (PAGE-Blot). An Enzyme-Linked-Imunosorbent Assay (ELISA) using mAb C4B6 has been developed, optimized and standardized. It has proven to be specific for DCP forms and has a minimum sensitivity of 0.156 A.U/ml.
The trimeric fiber of adenovirus type 2 (Ad2) mediates the first stage of virus-cell attachment, and the distal head region of the fiber has been implicated as the receptor-binding domain. To locate regions on the primary polypeptide sequence of the fiber which may be involved in virus-cell interaction, peptide-based epitope mapping was performed using (1) polyclonal antibodies prepared against both native Ad2 fiber and Ad2 head protein expressed in Escherichia coli and (2) 18 monoclonal antibodies prepared against trimeric Ad2 head protein expressed in baculovirus. The approach using polyclonal antibodies revealed eight domains on the primary sequence of the head which contain one or more continuous epitopes. At least two of these regions were also recognized by monoclonal antibodies reacting against both monomeric and trimeric fiber head protein. The majority of monoclonal antibodies which did not recognize Ad2 head-specific peptides in ELISA were also nonreactive against the monomeric form of protein in Western blot, suggesting that their recognition of trimer is due to the existence of as yet undefined discontinuous epitopes or to alterations in monomer configuration. Our results correspond well with the recently published X-ray crystallographic model of Ad5 fiber head (D. Xia, L.J. Henry, R.D. Gerard, and J. Deisenhofer, Structure 2, 1259-1270, 1994), since most antigenic determinants containing linear epitopes mapped to the outer loops or uppermost beta-sheets in this structure. Four of five neutralizing monoclonal antibodies recognized trimer only and none recognized linear peptides. This might suggest that the trimeric form of fiber is necessary for making contact with the receptor(s) and that discontinuous epitopes on the head domain may be involved in fiber-cell interaction.
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