Mouse Anti-EBNA-1 Recombinant Antibody (clone 3D4) (CAT#: FAMAB-0523WJ)

Figure 1 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.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 2 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.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 3 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.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 4 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.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 5 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.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 6 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.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 7 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.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 8 Binding of 3D4 to several antigens is examined by ELISA.

3D4 does not show significant binding to other antigens tested.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 9 Binding of 0211 to several antigens is examined by ELISA.

0211 binds moderately to Sm but not to the other antigens tested.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 10 3D4 binds to the carboxyl region while 0211 binds to both the carboxyl and amino regions of EBNA-1.

(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.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 11 3D4 recognizes a 148 aa core domain in the carboxyl region of EBNA-1.

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.

Yadav, P., Tran, H., Ebegbe, R., Gottlieb, P., Wei, H., Lewis, R. H., ... & Spatz, L. (2011). Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. PLoS One, 6(1), e14488.

Figure 12 IgG antibody to EBNA‐1 cross‐reacts with dsDNA.

IgG MAb, 3D4 binds strongly to EBNA-1 (A) and cross-reacts with dsDNA (B) as determined by ELISA. ELISAs are representative of three experiments. ELISA values were obtained in triplicate. Error bars indicate standard deviations of triplicates.

Yadav, P., Carr, M. T., Yu, R., Mumbey‐Wafula, A., & Spatz, L. A. (2016). Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA. Immunity, inflammation and disease, 4(3), 362-375.

Figure 13 Comparison of the binding of 3D4 and 16D2 to EBNA‐1.

The binding of 16D2 and 3D4 to EBNA-1were compared by ELISA. Goat anti-mouse kappa-AP was used as secondary antibody to detect the binding of 16D2 (an IgM MAb) and 3D4 (an IgG MAb) in the same assay. Results represent the average of two experiments. Standard deviation is indicated by error bars.

Yadav, P., Carr, M. T., Yu, R., Mumbey‐Wafula, A., & Spatz, L. A. (2016). Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA. Immunity, inflammation and disease, 4(3), 362-375.

Figure 14 Peptide mapping. 3D4 was tested by ELISA for binding to peptides spanning the entire 148 amino acid VBS region.

Yadav, P., Carr, M. T., Yu, R., Mumbey‐Wafula, A., & Spatz, L. A. (2016). Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA. Immunity, inflammation and disease, 4(3), 362-375.

Figure 15 3D4 was examined by ELISA for binding to GC‐15, GL‐15, and PFM‐15.

3D4 was used at a concentration of 5 μg/ml for all above ELISAs. ELISAs are representative of two experiments. ELISA values were obtained in triplicate.

Yadav, P., Carr, M. T., Yu, R., Mumbey‐Wafula, A., & Spatz, L. A. (2016). Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA. Immunity, inflammation and disease, 4(3), 362-375.

Figure 16 Peptide inhibition of MAbs binding to EBNA‐1 and dsDNA.

3D4 (0.05 μg/ml) were incubated with increasing concentrations of peptides PFM-15 or GC-15 and examined by ELISA for binding to EBNA-1 (B). 3D4 (2.5 μg/ml) were incubated with increasing concentrations of peptides PFM-15 or GC-15 and examined by ELISA for binding dsDNA (D).

Yadav, P., Carr, M. T., Yu, R., Mumbey‐Wafula, A., & Spatz, L. A. (2016). Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA. Immunity, inflammation and disease, 4(3), 362-375.

Figure 17 MAbs that bind to the carboxyl region of EBNA‐1 but do not cross‐react with dsDNA, recognize a different epitope than the cross‐reactive antibodies.

(A) The IgG isotype of the non-cross-reactive MAbs 2F8, 14E10, 4F6, and 4B8 were determined by ELISA. (B) The non cross-reactive MAbs, were tested by ELISA at a range of concentrations, for binding to EBNA-1. 3D4 was used as the reference standard. MAbs were tested by ELISA for binding to dsDNA (C), to the amino and carboxyl regions of EBNA-1 (D) and to two truncated carboxyl fragments; aa 452-641 and aa 459-607 (E). MAb concentrations used were 12.5 μg/ml in (C) and 1.25 μg/ml in (D) and (E). ELISAs are representative of two experiments. ELISA values were obtained in triplicate. Error bars represent the standard deviations of triplicates.

Yadav, P., Carr, M. T., Yu, R., Mumbey‐Wafula, A., & Spatz, L. A. (2016). Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA. Immunity, inflammation and disease, 4(3), 362-375.