Antigen-binding properties of monoclonal antibodies reactive with EBNA1 and use in immunoaffinity chromatography

Abstract

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs) were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG(1)) and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6) fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR)-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1.

Figure 1. Epitope mapping of the N-terminal anti-EBNA1 mAbs.

A) The constructs used to analyze mAb reactivity. Full length EBNA1 is shown at the top, numbered according to the B95-8 EBV strain. LR-1 and LR-2 (linking regions 1 & 2) are designated, as well as the GGA repeat region between amino acids 90 and 325. The C-terminus is largely composed of the DNA-binding and dimerization domain. 1553 encodes functionally wildtype EBNA1, but lacks the majority of the GGA stretch. The remaining EBNA1 derivatives are derived from the 1553 construct and thus carry the GGA deletion. B) 1891 was transfected into 293 cells and whole cell extract was analyzed by Western blot. The first lane in each series contains purified 1553, the second lane is untransfected 293 whole cell extract (mock), and the third lane is whole cell extract from cells transfected with the 1891 plasmid. Equal concentrations of purified mAbs were used to probed the Western blots. C) Plasmids 2728 and 2729 were transfected into 293 cells and whole cell extract was analyzed by Western blot. NT, not transfected. Equal concentrations of purified mAbs were used to probe the Western blots. D) Purified EBNA1 derivatives were analyzed by Western blot for mAb reactivity. Equal concentrations of mAb 1EB12 or mAb 1EB14 were used to probe the Western blots. E) Summary of the anti-EBNA1 mAb epitopes. The schematic shows endogenous EBNA1 protein, numbered according to the B95-8 strain of EBV, and the domain structure. The lines represent the various epitope regions and the mAbs that interact in that area are written below. MW is molecular weight.

Figure 2. Ability of mAbs to bind EBNA1/DNA complex.

MAbs were tested by EMSA to determine binding efficiency to a pre-formed EBNA1/DNA complex. Purified EBNA1 protein was bound to a ³²P-labelled oligonucleotide encoding one palindromic EBNA1 binding site. Equal concentrations of purified mAb were added to each reaction and allowed to incubate. The ability of the mAbs to shift the EBNA1/DNA complex was analyzed by gel electrophoresis. The first lane does not include any protein. The second lane contains only DNA and EBNA1. MAbs are listed by the last number in their name.

Figure 3. Immunoprecipitation of EBNA1.

E. coli-expressed EBNA1 (∼50 kDa) was isolated from whole cell extract by mixing with equal quantities of mAb-rProteinA-agarose resin. Reactions were allowed to incubate for 1 h, washed to remove nonspecifically bound proteins, and eluted with SDS sample buffer. MW is molecular weight. The Western blot was probed with rat mAb 2B4 .

Figure 4. Polyol-responsiveness of anti-EBNA1 mAbs.

A) ELISA-elution assay results to determine polyol-responsiveness. A standard ELISA protocol was performed with the addition of an intermediate step. After the anti-EBNA1 mAbs are bound to the EBNA1-coated well, either a control buffer (TE: 50 mM Tris-HCl, pH 7.9+0.1 mM EDTA) or a salt-polyol buffer (AS/PG: TE buffer+1 M ammonium sulfate+40% propylene glycol or NaCl/PG: TE buffer+1 M NaCl+40% propylene glycol) was added to the wells. MAbs that are polyol-responsive will have lower affinity for their antigen in the salt-polyol buffer which will result in a decrease in signal after the addition of a secondary antibody and the ELISA substrate. The experiment was done in triplicate. B) Detailed analysis of mAb 3EB7 response to various combinations of ammonium sulfate (AS) and propylene glycol.

Figure 5. Immunoaffinity chromatography using PR-mAbs.

The soluble fraction of whole cell extract from E. coli overexpressing EBNA1 was added to A) mAb 1EB2-Sepharose column and B) mAb 3EB7-Sepharose column, washed, and eluted with TE+0.75 M AS+40% PG. Coomassie blue stained gels are shown.