Serodiagnosis of Lyme borreliosis infection using surface plasmon resonance.

BACKGROUND: Label-free biosensors are ideally suited for the direct monitoring of binding events without the need for additional labeling substances; however, their application in the field of serodiagnosis is not trivial. The major problem is the unspecific adsorption of blood serum components to the sensor surface. METHODS: A surface plasmon resonance (SPR) sensor has been used for the direct detection of Lyme borreliosis specific antibodies in blood serum. The combination of an optimal dilution factor with the addition of suitable detergents minimizes the unspecific adsorption. Serum samples from healthy donors and infected patients have been analyzed and the results were compared with a certified immunoassay and a western blot. RESULTS: A serum dilution of 1:20 in HBS-buffer with 0.05% Tween20 and 1 mg/mL carboxymethyl dextran reduces unspecific adsorption significantly and enables the identification of antibodies against the OspC/pepC10 antigen pair with a sensitivity of 92% and that against the VlsE/C6 pair with 81% sensitivity; the specificities are 82% and 86% respectively. Positive hits in the western blot could also be determined in the SPR-assay with a correlation of 96.5%. CONCLUSION: The presented optical label-free technique has the potential for a precise and fast identification of pathogen-specific antibodies without the need for a secondary labeling antibody.

Application of an electric DNA-chip for the expression analysis of bioprocess-relevant marker genes of Bacillus subtilis.

The knowledge of critical process-relevant genes can be used for an improved control of bioprocesses. So far bioprocess-relevant marker genes can be analyzed by established expression analysis methods only off-line. In this study, an alternative approach for a potential at-line monitoring of gene expression during bioprocesses is suggested. This approach is based on the measurement of specific mRNAs on an electric DNA-chip in connection with a magnetic bead-based sandwich hybridization. In order to allow an at-line measurement of specific mRNAs an improved method for a fast and partially automated isolation of high quality-RNA samples was developed. The expression analysis of the electric DNA-chip was compared with optical DNA micro arrays and the real time RT-PCR for three selected process-relevant genes of Bacillus subtilis. We demonstrate that the mRNA analysis by means of the electric DNA-chip gives similar results compared to the micro array analysis and the real time RT-PCR technique.

Global expression profiling of Bacillus subtilis cells during industrial-close fed-batch fermentations with different nitrogen sources.

A detailed gene expression analysis of industrial-close Bacillus subtilis fed-batch fermentation processes with casamino acids as the only nitrogen source and with a reduced casamino acid concentration but supplemented by ammonia was carried out. Although glutamine and arginine are supposed to be the preferred nitrogen sources of B. subtilis, we demonstrate that a combined feeding of ammonia and casamino acids supports cell growth under fed-batch fermentation conditions. The transcriptome and proteome analyses revealed that the additional feeding of ammonia in combination with a reduced amino acid concentration results in a significantly lower expression level of the glnAR or tnrA genes, coding for proteins, which are mainly involved in the nitrogen metabolism of B. subtilis. However, the mRNA levels of the genes of the ilvBHC-leuABD and hom-thrCB operons were significantly increased, indicating a valine, leucine, isoleucine, and threonine limitation under these fermentation conditions. In contrast, during the fermentation with casamino acids as the only nitrogen source, several genes, which play a crucial role in nitrogen metabolism of B. subtilis (e.g., glnAR, nasCDE, nrgAB, and ureABC), were up-regulated, indicating a nitrogen limitation under these conditions. Furthermore, increased expression of genes, which are involved in motility and chemotaxis (e.g., hag, fliT) and in acetoin metabolism (e.g., acoABCL), was determined during the fermentation with the mixed nitrogen source of casamino acids and ammonia, indicating a carbon limitation under these fermentation conditions. Under high cell density and slow growth rate conditions a weak up-regulation of autolysis genes could be observed as well as the induction of a number of genes involved in motility, chemotaxis and general stress response. Results of this study allowed the selection of marker genes, which could be used for the monitoring of B. subtilis fermentation processes. The data suggest for example acoA as a marker gene for glucose limitation or glnA as an indicator for nitrogen limitation.