Figure 1.
rEBNA-1 injected mice develop antibodies to EBNA-1 and dsDNA.
Mice were unimmunized or injected ip with rEBNA-1 emulsified in CFA or CFA alone and boosted with rEBNA-1 emulsified in IFA or IFA alone (arrows indicate week of initial injection and boosts). Mice were bled at weeks 1.5, 4, 6, 10, 12, 15, and 18 and sera tested by ELISA for antibody to EBNA-1 (A) and dsDNA (B). Results are the average of 5 mice in each group. Standard deviations are indicated. (C and D) Eight fold serial dilutions of week 12 sera from all 5 rEBNA-1 injected mice were tested by ELISA for antibody titers to EBNA-1 (C) and dsDNA (D). Dotted line represents 3 standard deviations above the mean absorption of sera from week 12 uninjected control mice. (E) Serum from a week 12 mouse injected with rEBNA-1 (left panel) or adjuvant only (right panel) was diluted 1∶50 and used to immunostain Crithidia luciliae slides. Immunostaining of the dsDNA in the kinetoplast of Crithidia luciliae is observed (left panel) as indicated by arrow. Results are representative of 3 rEBNA-1 and 3 adjuvant only, injected mice.
Figure 2.
Sera from EBNA-1 injected mice display reduced binding to EBNA-1 following adsorption onto dsDNA cellulose beads.
Week 12 sera from all five rEBNA-1 injected mice were adsorbed onto dsDNA cellulose beads and pre and post adsorbed sera were tested by ELISA for binding to dsDNA (A) and EBNA-1 (B). Results represent OD 405 nm values after subtraction of non specific binding to cellulose only beads. Standard deviations of triplicate wells are indicated. Sera were diluted 1∶5000 prior to adsorption. One tailed, unpaired t test was used to compare OD 405 nm before and after adsorption on dsDNA cellulose (t<0.001 for mouse 1,3, 4, and 5 in (A) and t<0 .005 for mouse 1, 3, 4, and 5 in (B)).
Table 1.
Reactivity of representative monoclonal antibodies to EBNA-1, dsDNA, and BSA.
Figure 3.
MAb, 3D4, is specific for both EBNA-1 and dsDNA.
(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 strep-tag and were detected with Strep-Tactin-HRP.
Figure 4.
Commercial MAb, 0211 cross-reacts with dsDNA.
(A) MAb 0211 binds to EBNA-1 as detected by ELISA but not P2, or BSA. (B) MAb 0211 binds to dsDNA as detected by ELISA. (C & D) MAb 0211 was adsorbed onto dsDNA cellulose beads and pre and post adsorbed antibody were tested for binding to dsDNA and EBNA-1 by ELISA. (C) MAb 0211 adsorbed on dsDNA cellulose beads, was completely depleted of antibody reactivity for dsDNA and EBNA-1. Anti-dsDNA and anti-EBNA-1 ELISAs were performed on separate ELISA plates and ELISAs were developed when ODs reached maximal values. Results represent OD 405 nm values after subtraction of non specific binding to cellulose only beads. (D) Post dsDNA cellulose adsorbed MAb 0211 shows reduced binding to rEBNA-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 0211 (lane 4) as indicated.
Figure 5.
Comparison of the binding affinity and specificity of 3D4 and 0211.
(A) Binding of 3D4 to EBNA-1 is compared to that of 0211 by ELISA at two different concentrations of MAbs. 3D4 binds more strongly to EBNA-1 than 0211. (B) Binding of 3D4 to several antigens is examined by ELISA. 3D4 does not show significant binding to other antigens tested. (C) Binding of 0211 to several antigens is examined by ELISA. 0211 binds moderately to Sm but not to the other antigens tested.
Figure 6.
3D4 binds to the carboxyl region while 0211 binds to both the carboxyl and amino regions of EBNA-1.
(A) Functional map of the complete EBNA-1 protein containing the Gly-Ala repeat region (GA). CBS 1,2,3; chromatin binding sites, NLS; nuclear localization site, VBS: viral binding site. LS8 denotes the amino fragment (aa 1–404) lacking most of the GA repeat. LS7 denotes the amino fragment (aa 1–393) lacking the GA repeat and lacking PPPGRRP. LS9 denotes the carboxyl fragment (aa 410–641). (B) MAb 3D4 is strongly reactive with LS9 but not LS8 or LS7 as detected by ELISA. (C) MAb 0211 is reactive with LS7, LS8 and LS9 as detected by ELISA. Results in B and C are the average of triplicate wells.
Figure 7.
3D4 recognizes a 148 aa core domain in the carboxyl region of EBNA-1.
(A) Map of the carboxyl region of EBNA-1 and 3 truncated carboxyl fragments. (B) 3D4 was tested by ELISA for binding to the 3 truncated fragments of EBNA-1. 3D4 binds strongly to all 3 fragments, the smallest being EBNA459–607.