We developed a quartz crystal biosensor designed to detect concentrations and ligand affinity parameters of free unlabeled proteins in real time. Using a model system with human IgE as the analyte and single-stranded DNA aptamers or an anti-IgE antibody as immobilized ligands, we could demonstrate that aptamers were equivalent to antibodies in terms of specificity and sensitivity. Both receptor types selectively detected 0.5 nmol/L of IgE. In addition, the aptamer receptors tolerated repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. Because of the small size and nonprotein nature of the aptamers, they were immobilized in a dense, well-oriented manner, thus extending the linear detection range to 10-fold higher concentrations of IgE. In addition to demonstrating for the first time that an aptamer-based biosensor can specifically and quantitatively detect an analyte in various complex protein mixes, the aptamer-ligand proved to be relatively heat resistant and stable over several weeks. Since aptamers consist of nucleic acids, well-established chemistry can be applied to produce optimized affine layers on biosensors that may be developed to specifically detect proteins in solution for analysis of proteomes.

The unique region of structural protein VP1 of parvovirus B19 (erythrovirus B19) is important for eliciting neutralizing antibodies that are responsible for eliminating the virus from the peripheral blood and for inducing lifelong immunity. Neutralizing human MAbs bind a conformationally defined epitope spanning VP1 residues 30-42. The DNA sequence encoding the VP1-unique region was determined in parvovirus B19 isolated from peripheral blood and amniotic fluid of nine acutely infected pregnant women, five arthritis patients and two chronically infected children. The amino acid sequences of the VP1-unique region exhibited higher variability in comparison with other B19-specific proteins. To analyse the influence of amino acid variations on antibody binding and protein conformation, two variants of the VP1-unique region were selected and expressed in E. coli as intein-fusion proteins. The selected variants displayed a number of amino acid exchanges in the VP1-unique region and had mutations in the determined epitope and adjacent regions. After purification via affinity chromatography, the dissociation constants K D of VP1-specific human MAbs interacting with the variant antigens and a viral prototype of the VP1-unique region were determined with a quartz crystal microbalance biosensor. A value of 5n4i10 N8 M was determined for the prototype isolate pJB ; the affinity constants for the variant VP1-unique regions were similar. Comparable values were obtained for interaction of antibodies with non-infectious VP1/VP2 capsids produced by recombinant baculovirus and with B19 virions from amniotic fluid. It is concluded that the conformation of the epitope is unaffected by mutations or the environment of the VP1-unique region in virus capsids.

The transformation of a panel of rpoB mutations (codons 525 to 586) from rifampin-resistant donor strains into Helicobacter pylori 2802A confirmed the MICs associated with the respective mutations. RpoB V149X random mutations were generated and induced different levels of resistance, depending on the replacement amino acid. Mutagenesis of the rpoB gene at codon position 701 (R701H) induced low-level resistance.Rifabutin and some other rifamycin derivatives inhibit the growth of Helicobacter pylori in vitro at much lower concentrations than rifampin and might be possible candidates for second-or third-line eradication therapy (1, 2). It has been shown that triple therapy containing rifabutin is effective in the eradication of H. pylori after failure of other therapies and in spite of resistance to other antibiotics (10).The target of all rifamycins is the ␤-subunit of the DNAdependent RNA polymerase encoded by the rpoB gene (4, 5). Amino acid substitutions resulting from changes in codons 507 to 533 of rpoB in mycobacteria (8, 9), codons 146, 507 to 534, 563 to 572, and 687 in Escherichia coli (4, 6, 11), and codons 473 to 483 and 528 to 530 in Listeria monocytogenes (7) induce resistance.Resistant mutants of H. pylori ATCC 43504 selected in vitro by serial passage in the presence of rifampin all showed mutations in codons 525 to 545 or codon 586 (2). Recently, we described a clinical isolate of H. pylori that developed resistance during therapy and that harbored a mutation at codon 149 (V149F), corresponding to the V146F mutation observed in E. coli (6). Consequently, we detected a homologous mutation (V176F) in three clinical isolates of Mycobacterium tuberculosis (3).In this study, we tried a compilation of possible mutations in H. pylori at four different regions of rpoB which are highly conserved between different species that are susceptible to rifamycins. Codon 149 was randomized by site-directed mutagenesis. Arginine 701, as encoded by E. coli codon 687, was replaced with histidine. The levels of resistance of these new mutants were determined and compared to the resistance levels of the panel of H. pylori ATCC 43504 variants with mutations in codons 525 to 545 (cluster I) or in codon 585 (cluster II).Culture, susceptibility testing, and transformation conditions were described recently (2, 3). H. pylori suspensions adjusted to McFarland 2 were tested with a semiautomatic multipoint inoculator (Multipoint AD; Mast) in the agar dilution assay. Rifabutin and rifampin concentrations ranged from 0.002 to 256 g/ml. Type strains were included for quality control.

No abstract