Display of a peptide mimotope on a crystalline bacterial cell surface layer (S-layer) lattice for diagnosis of Epstein-Barr virus infection.
Fusion proteins based on the crystalline bacterial cell surface layer (S-layer) proteins SbpA from Bacillus sphaericus CCM 2177 and SbsB from Geobacillus stearothermophilus PV72/p2 and a peptide mimotope F1 that mimics an immunodominant epitope of Epstein-Barr virus (EBV) were designed and overexpressed in Escherichia coli. Constructs were designed such that the peptide mimotope was presented either at the C-terminus (SbpA/F1) or at the N-terminus (SbsB/F1) of the respective S-layer proteins. The resulting S-layer fusion proteins, SbpA/F1 and SbsB/F1, fully retained the intrinsic self-assembly capability of the S-layer moiety into monomolecular lattices. As determined by immunodot assays and ELISAs using monoclonal antibodies, the F1 mimotope was well-presented on the outer surface of the S-layer lattices and accessible for antibody binding. Further comparison of the two S-layer fusion proteins showed that the S-layer fusion protein SbpA/F1 had a higher antibody binding capacity than SbsB/F1 in aqueous solution and in immune sera, illustrating the importance of epitope orientation on the performance of solid-phase immunoassays. To assess the diagnostic values of S-layer mimotope fusion protein SbpA/F1, we screened a panel of 83 individual EBV IgM-positive, EBV negative, and potential cross-reactive sera for their reactivities. This resulted in 98.2% specificity and 89.3% sensitivity, and furthermore no cross-reactivity with related viral disease states including rheumatoid factor was observed. This study shows the potential of S-layer fusion proteins as a matrix for site-directed immobilization of small ligands in solid-phase immunoassays using EBV diagnostics as a model system.
Tschiggerl
Helga
H
Center for NanoBiotechnology, University of Natural Resources and Applied Life Sciences, Gregor-Mendel-Strasse 33, A-1180 Vienna, Austria.
Casey
Joanne L
JL
Parisi
Kathy
K
Foley
Michael
M
Sleytr
Uwe B
UB
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D040761:Biomimetic Materials / Q000737:chemistry / Q000378:metabolism; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D020031:Epstein-Barr Virus Infections / Q000175:diagnosis* / Q000276:immunology; D015870:Gene Expression; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity
D016056:Immunodominant Epitopes; D008562:Membrane Glycoproteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins