The LabTube - a novel microfluidic platform for assay automation in laboratory centrifuges.

Assay automation is the key for successful transformation of modern biotechnology into routine workflows. Yet, it requires considerable investment in processing devices and auxiliary infrastructure, which is not cost-efficient for laboratories with low or medium sample throughput or point-of-care testing. To close this gap, we present the LabTube platform, which is based on assay specific disposable cartridges for processing in laboratory centrifuges. LabTube cartridges comprise interfaces for sample loading and downstream applications and fluidic unit operations for release of prestored reagents, mixing, and solid phase extraction. Process control is achieved by a centrifugally-actuated ballpen mechanism. To demonstrate the workflow and functionality of the LabTube platform, we show two LabTube automated sample preparation assays from laboratory routines: DNA extractions from whole blood and purification of His-tagged proteins. Equal DNA and protein yields were observed compared to manual reference runs, while LabTube automation could significantly reduce the hands-on-time to one minute per extraction.

Evaluation of three rapid assays for detection of Clostridium difficile toxin A and toxin B in stool specimens.

Diagnosis of Clostridium difficile-associated disease continues to be difficult for clinical microbiology laboratories. The aim of this study was to evaluate the performance of three enzyme immunoassays for detection of C. difficile toxins A and B: the recently marketed rapid enzyme immunoassay Ridascreen Clostridium difficile Toxin A/B (R-Biopharm, Darmstadt, Germany) and two established enzyme immunoassays, the C. difficile Tox A/B II Assay (TechLab, Blacksburg, VA, USA) and the ProSpecT C. difficile Toxin A/B Microplate Assay (Remel, Lenexa, KS, USA). Stool specimens (n = 383) from patients with a clinical diagnosis of antibiotic-associated diarrhea were examined by these three enzyme immunoassays and were additionally cultured for C. difficile on selective agar. Samples giving discordant enzyme immunoassay results underwent confirmatory testing by tissue culture cytotoxin B assay and by PCR for toxin A (tcdA) and toxin B (tcdB) genes from C. difficile. Using the criteria adopted for this study, 60 (15.7%) samples tested positive for toxins A and/or B. Sensitivity and specificity of the enzyme immunoassays were, respectively, 88.3 and 100% for the TechLab enzyme immunoassay, 91.7 and 100% for the R-Biopharm enzyme immunoassay, and 93.3 and 100% for the Remel enzyme immunoassay. The differences between these results are statistically not significant (p > 0.05). The results show that all three enzyme immunoassays are acceptable tests for the detection of C. difficile toxins A and B directly in fecal specimens or in toxigenic cultures.

Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation.

BACKGROUND: The development of macrolide resistance in Helicobacter pylori is considered an essential reason for failure of antibiotic eradication therapies. The predominant mechanism of resistance to macrolides, particularly clarithromycin, is based on three defined mutations within 23S rRNA, resulting in decreased binding of the antibiotic to the bacterial ribosome. AIM: To develop an rRNA based whole cell hybridisation method to detect Helicobacter species in situ within gastric tissue, simultaneously with its clarithromycin resistance genotype. METHODS: A set of fluorescent labelled oligonucleotide probes was developed, binding either to H pylori 16S rRNA or 23S rRNA sequences containing specific point mutations responsible for clarithromycin resistance. After hybridisation and stringent washing procedures, labelling of intact single bacteria was monitored by fluorescence microscopy. The new approach was compared with PCR based assays, histology, and microbiological culture. RESULTS: In comparison with the phenotypic resistance measurement by E test, the genotypic clarithromycin resistance correlated perfectly (100%) for 35 H pylori isolates analysed. In a set of gastric biopsy specimens (27) H pylori infection was confirmed by histology (17/27) and correctly detected by whole cell hybridisation. Five clarithromycin resistant strains were identified in gastric tissue specimens directly. Furthermore, non-cultivable coccoid forms of H pylori were easily detectable by whole cell hybridisation. CONCLUSIONS: Whole cell hybridisation of rRNA holds great promise for cultivation independent, reliable, and rapid (three hours) genotypic determination of clarithromycin resistance in H pylori. Compared with PCR techniques it is independent of nucleic acid preparations, not prone to inhibition, and allows semiquantitative visualisation of the bacteria within intact tissue samples.

T cell cross-reactivity to heavy metals: identical cryptic peptides may be presented from protein exposed to different metals.

The mechanisms by which metals induce activation of T cells and thus produce allergic and/ or autoimmune reactions are still obscure, and the same is true for the mechanisms that underly T cell cross-reactivity to different heavy metal ions. In the present study, we investigated induction by metals of T cell reactions to cryptic peptides of bovine RNase A. Murine CD4+ T cell hybridomas specific for cryptic RNase peptides presented from Au(III)-treated RNase were used as detection probes. We showed that in vitro treatment of RNase with Pd(II), Pd(IV), Ni(IV), and partially Pt(IV), but not Au(I), Ni(II), or Pt(II), induced presentation of the same cryptic peptides as those presented from Au(III)-treated RNase. That the former heavy metal ions, but not the latter, were able to alter the antigenicity of RNase was reflected by their ability to induce conformational changes of RNase, as detected by circular dichroism spectroscopy. Furthermore, upon immunization against RNase pretreated with these metals, CD4+ T cell hybridomas specific for unidentified cryptic peptides were obtained. In conclusion, "metal-specific" T cell reactions may be directed against cryptic peptides, and metal cross-reactivity in allergic individuals might be due to metal-induced presentation of overlapping, but not identical, panels of cryptic peptides.

Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B.

Helicobacter pylori is a Gram-negative bacterial pathogen associated with gastritis, peptic ulceration, and gastric carcinoma. The bacteria express a strong urease activity which is known to be essential for colonization of gnotobiotic pigs and nude mice. UreA and UreB, two structural subunits of the active enzyme, were expressed in the attenuated Salmonella typhimurium live vaccine SL3261 strain. Evaluation of protection against H. pylori was performed in Balb/c mice by oral immunization with a single dose of the vaccine strain. Five weeks after immunization, mice were challenged orally three times with a mouse-adapted H. pylori wild type strain and, six weeks later, mice were sacrificed to determine H. pylori infection by detection of urease activity from the antral region of the mouse stomachs. In several independent experiments, we observed 100% infection with H. pylori in the non-immunized mice and no infection (100% protection) in the mice immunized with S. typhimurium expressing recombinant UreA and UreB. Specific humoral and mucosal antibody responses against UreA and UreB were observed in mice immunized as indicated by western blots and ELISA assays. These data shows that oral immunization of mice with urease subunits delivered by an attenuated Salmonella strain induced a specific immune response and protected mice against H. pylori colonization. Single oral dose immunization with UreA and UreB delivered by a live Salmonella vaccine vector appears to be an attractive candidate for human vaccination against H. pylori infection. In addition, this model will aid to elucidate the effective protection mechanisms against H. pylori in the gastric mucosa.

Alteration of a model antigen by Au(III) leads to T cell sensitization to cryptic peptides.

Certain metal ions are known to be potent sensitizers, but the self proteins modified by metal ions and the self peptides recognized by 'metal-specific' T cells are unknown. In humans and mice treatment with gold anti-rheumatic drugs, containing Au(I), may lead to allergic and autoimmune side effects. Human and murine T cells do not react to Au(I), however, but to the reactive metabolite Au(III). Here we show that alteration by Au(III) of a model antigen, bovine ribonuclease (RNase)A, results in T cell sensitization to cryptic peptides of this protein. Upon immunization of mice with Au(III)-pretreated RNase [RNase/Au(III)], CD4+ T cell hybridomas specific for RNase/Au(III) were obtained in addition to those recognizing the immunodominant peptide RNase 74-88; the latter also were obtained after immunization with native RNase. RNase/Au(III)-specific T cell hybridomas reacted against RNase/Au(III) and RNase denatured by S-sulfonation of cysteine residues, but not against native RNase, or RNase pretreated with Au(I), A1(III), Cu(II), Fe(II), Fe(III), Ni(II), Mn(II), or Zn(II). Using a panel of overlapping, synthetic RNase peptides which were devoid of gold or gold-induced modifications, epitope mapping revealed that RNase/Au(III)-specific T cell hybridomas recognized the cryptic peptides 7-21 and 94-108, respectively. Comparison of the proliferative response of bulk CD4+ T cells, prepared from splenocytes after immunization with either RNase/Au(III) or native RNase, revealed that Au(III) pretreatment of RNase led to a markedly enhanced response to the two cryptic peptides while it did not influence the response to the immunodominant peptide. The cryptic peptides were also presented after preincubation of bone marrow-derived macrophages with RNase and Au(I), but not with RNase alone, suggesting that oxidation of Au(I) to Au(III) and subsequent protein alteration by Au(III) can happen in mononuclear phagocytes. We conclude that Au(III) alteration of proteins alters antigen processing and, thus leads to presentation of cryptic peptides. This mechanism may shed light on the development of allergic and autoimmune side effects of Au(I) anti-rheumatic drugs. In addition, it might provide a general mechanism of how metal ions act as T cell sensitizers.