Immobilized stem–loop structured probes as conformational switches for enzymatic detection of microbial 16S rRNA

We have designed and evaluated novel DNA stem–loop structured probes for enzymatic detection of nucleic acid targets. These probes constitute a novel class of conformational switches for enzymatic activity, which in the absence of a target sterically shield an affinity label and upon hybridization of the target to the recognition sequence that forms the loop of the probe restore accessibility of the label for the binding of a reporter enzyme. Analysis of probe characteristics revealed stem stability as the most important parameter governing detection functionality, while other factors such as the length of linker molecules attaching the label to the stem–loop structure and the nature of the solid support proved to be less critical. Apparently, the bulky nature of the reporter enzyme facilitates shielding of the label in the absence of the target, thereby conferring considerable structural tolerance to the conformational switch system. The stem–loop structured probes allow sensitive detection of unlabeled nucleic acid targets. Employing a microtiter assay format, 4 ng of bacterial 16S ribosomal RNA corresponding to 8 fmol could be detected, which can be compared favorably with current immobilized molecular beacon concepts based on fluorescence detection

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity

<p>Abstract</p> <p>Background</p> <p>Hymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far.</p> <p>Methods</p> <p>Expression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients.</p> <p>Results</p> <p>Both major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients.</p> <p>Conclusions</p> <p>The recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy.</p

Complement Inactivation by Recombinant Human C3 Derivatives

From the implications of the complement system in a large number of diseases, an urgent need for therapeutics effecting reduced complement activity in vivo has emerged. In this study we report the design of a novel class of enzymes of human origin that obliterate functional complement by a noninhibitory, catalytic mechanism. Combining the framework of human C3 and the enzymatic mechanism of cobra venom factor, a nontoxic snake venom protein, we established molecules capable of forming stable C3 convertase complexes. Although the half-life of naturally occurring C3 convertase complexes ranges between 1 and 2 min, these complexes exhibit a half-life of up to several hours. Because the overall identity to human C3 could be extended to >90%, the novel C3 derivatives can be assumed to exhibit low immunogenicity and, therefore, represent promising candidates for therapeutic reduction of complement activity in vivo.PeerReviewe

Structure of linker molecules employed for biotin attachment to the stem–loop structures

<p><b>Copyright information:</b></p><p>Taken from "Immobilized stem–loop structured probes as conformational switches for enzymatic detection of microbial 16S rRNA"</p><p>Nucleic Acids Research 2005;33(11):e101-e101.</p><p>Published online 24 Jun 2005</p><p>PMCID:PMC1159122.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> ARP-biotin [-(aminooxyacetyl)--(-biotinoyl)hydrazine] is attached via Schiff base chemistry to an aldehyde group generated by oxidative cleavage of the terminal ribose moiety of cytidin (rC). C3-Biotin contains a C3-ether-glycerol linker attached to the biotin residue via an amide bond. In TEG-biotin, the C3-ether-glycerol linker is extended by a triethyleneglycol (TEG) moiety

Quantitation of serum IgE by using chimeras of human IgE receptor and avian immunoglobulin domains

Anti-IgE therapeutics represent an efficient approach in the management of IgE-mediated allergic asthma. However, monitoring the reduction of IgE levels into a therapeutically efficient range requires the determination of residual serum IgE. We established an analytical approach to distinguish free and anti-IgE complexed serum IgE based on soluble derivatives of the human high-affinity IgE receptor. Soluble receptor derivatives represent an ideal means to analyze receptor antagonism by any ligand or blocking antibody. Therefore, the FcRI ectodomain was fused with avian IgY constant domains that circumvent susceptibility to interference phenomena and improve assay performance. After production in HEK293 cells, subsequent characterization by enzyme-linked immunosorbent assay and immunoblotting confirmed the suitability of avian IgY constant domains for immobilization and detection purposes. To provide further insights into the different IgE reactivities, free allergen-specific IgE was also determined. Monitoring of sera from omalizumab-treated patients during the course of therapy revealed the applicability for assessment of omalizumab-complexed versus noncomplexed serum IgE. These parameters may allow correlation to clinical responses during anti-IgE therapy with the perspective of biomonitoring. © 2011 Elsevier Inc. All rights reserved