MAb, 3D4, is specific for both EBNA-1 and dsDNA.

<p>(A) Anti-EBNA-1 ELISA. 3D4 was tested by ELISA, at increasing concentrations, for binding to EBNA-1, BSA, and the cystovirus, polymerase protein, P2 (negative control) coated on Costar plates. Plates were read at 405 nm at 20 minutes. 3D4 shows specificity for EBNA-1 under these conditions. Results are the averages of triplicates and standard deviations are indicated. (B) 3D4 was tested by ELISA for binding to dsDNA coated on Immulon-2 plates. Plates were read at 405 nm at 1 hour. Results in A and B are the average of triplicates and standard deviations are indicated. (C) 3D4 antibody is of the IgG1 isotype. ELISA plates coated with unlabeled anti-IgG1, anti-IgG2a, anti-IgG2b, or anti-IgG3 were incubated with 1.5 µg/ml of 3D4 MAb followed by the respective polyclonal isotype specific antibodies conjugated to alkaline phosphatase. (D) Purified 3D4 at a concentration of 10 µg/ml was examined for binding to Crithidia luciliae slides. Left panel shows the binding of 3D4 to kinetoplasts of Crithidia luciliae (arrow). Right panel; IgG1 isotype control antibody, does not bind specifically to kinetoplasts. (E & F) 3D4 was adsorbed on a dsDNA cellulose column and pre and post adsorbed antibody were tested for binding to dsDNA and EBNA-1 by ELISA (E) and to EBNA-1 by Western blot (F). (E) 3D4 adsorbed on dsDNA cellulose beads was completely depleted of antibody with reactivity for dsDNA and EBNA-1 as detected by ELISA. Results represent OD 405 nm values after subtraction of non specific binding to cellulose only beads. Standard deviations of triplicate wells are indicated. Anti-dsDNA and anti-EBNA-1 ELISAs were performed on Immulon-2 and Costar plates respectively and ELISAs were developed when ODs on each plate reached maximal values. (F) Post dsDNA cellulose adsorbed 3D4 shows reduced binding to EBNA-1 by Western blot. Left panel: Coomassie stained polyacrylamide gel. Right panel: Western blot: filters were immunostained with pre (lanes 2 and 3) or post dsDNA cellulose adsorbed 3D4 MAb (lane 4). Molecular weight markers used in Western blot were conjugated to <i>strep-tag</i> and were detected with Strep-Tactin-HRP.