Ultraviolet C inactivation of Coxiella burnetii for production of a structurally preserved whole cell vaccine antigen.

Q fever, a worldwide-occurring zoonotic disease, can cause economic losses for public and veterinary health systems. Vaccines are not yet available worldwide and currently under development. In this regard, it is important to produce a whole cell antigen, with preserved structural and antigenic properties and free of chemical modifications. Thus, inactivation of Coxiella burnetii with ultraviolet light C (UVC) was evaluated. C. burnetii Nine Mile phase I (NMI) and phase II (NMII) were exposed to decreasing intensities in a time-dependent manner and viability was tested by rescue cultivation in axenic medium or cell culture. Effects on the cell structure were visualized by transmission electron microscopy and antigenicity of UVC-treated NMI was studied by immunization of rabbits. NMI and NMII were inactivated at UVC intensities of 250 µW/cm2 for 5 min or 100 µW/cm2 for 20 min. Reactivation by DNA repair was considered to be unlikely. No morphological changes were observed directly after UVC inactivation by transmission electron microscopy, but severe swelling and membrane degradation of bacteria with increasing severity occurred after 24 and 48 h. Immunization of rabbits resulted in a pronounced antibody response. UVC inactivation of C. burnetii resulted in a structural preserved, safe whole cell antigen and might be useful as antigen for diagnostic purposes or as vaccine candidate.

A rapid test recognizing mucosal SARS-CoV-2-specific antibodies distinguishes prodromal from convalescent COVID-19.

The COVID-19 pandemic poses enormous challenges to global healthcare sectors. To prevent the overburden of medical systems, it is crucial to distinguish individuals approaching the most infectious early phase from those in the declining non-infectious phase. However, a large fraction of transmission events occur during pre- or asymptomatic phases. Especially in the absence of symptoms, it is difficult to distinguish prodromal from late phases of infection just by RT-PCR since both phases are characterized by low viral loads and corresponding high Ct values (>30). We evaluated a new rapid test detecting IgG antibodies recognizing SARS-CoV-2 nucleocapsid protein using two commercial antibody assays and an in-house neutralization test before determining suitability for testing clinical swab material. Our analyses revealed the combination of the well-known RT-PCR and the new rapid antibody test using one single clinical nasopharyngeal swab specimen as a fast, cost-effective, and reliable way to discriminate prodromal from subsiding phases of COVID-19.

Evaluation of the Diagnostic Potential of Recombinant Coxiella burnetii Com1 in an ELISA for the Diagnosis of Q Fever in Sheep, Goats and Cattle.

Coxiella burnetii is the causative agent of Q fever, a zoonosis infecting domestic ruminants and humans. Currently used routine diagnostic tools offer limited sensitivity and specificity and symptomless infected animals may be missed. Therefore, diagnostic tools of higher sensitivity and specificity must be developed. For this purpose, the C. burnetii outer membrane protein Com1 was cloned and expressed in Escherichia coli. The His-tagged recombinant protein was purified and used in an indirect enzyme-linked immunosorbent assay (ELISA). Assay performance was tested with more than 400 positive and negative sera from sheep, goats and cattle from 36 locations. Calculation of sensitivity and specificity was undertaken using receiver operating characteristic (ROC) curves. The sensitivities and specificities for sheep were 85% and 68% (optical density at 450nm, OD450 cut-off value 0.32), for goats 94% and 77% (OD450 cut-off value 0.23) and for cattle 71% and 70% (OD450 cut-off value 0.18), respectively. These results correspond to excellent, outstanding and acceptable discrimination of positive and negative sera. In summary, recombinant Com1 can provide a basis for more sensitive and specific diagnostic tools in veterinary medicine.

Rapid and sensitive point-of-care detection of Orthopoxviruses by ABICAP immunofiltration.

BACKGROUND: The rapid and reliable detection of infectious agents is one of the most challenging tasks in scenarios lacking well-equipped laboratory infrastructure, like diagnostics in rural areas of developing countries. Commercially available point-of-care diagnostic tests for emerging and rare diseases are particularly scarce. RESULTS: In this work we present a point-of-care test for the detection of Orthopoxviruses (OPV). The OPV ABICAP assay detects down to 1 × 104 plaque forming units/mL of OPV particles within 45 min. It can be applied to clinical material like skin crusts and detects all zoonotic OPV infecting humans, including Vaccinia, Cowpox, Monkeypox, and most importantly Variola virus. CONCLUSIONS: Given the high sensitivity and the ease of handling, the novel assay could be highly useful for on-site diagnostics of suspected Monkeypox virus infections in areas lacking proper laboratory infrastructure as well as rapid on-site testing of suspected bioterrorism samples.

Rapid flow through immunoassay for CRP determination based on polyethylene filters modified with ω-aminocellulose carbamate.

A novel method for antibody immobilization is discussed and applied in a column based flow through immunoassay system (ABICAP). It is shown that porous polyethylene material can be modified with different ω-aminocellulose carbamates yielding an amino group containing biocompatible support for antibody immobilization. Anti-h CRP antibodies can be bound covalently to the surface via various homobifunctional cross-linkers. The antibody modified filters are validated for the CRP determination in a sandwich ABICAP system. In a 10 min test procedure based on colloidal dye particles, a limit of detection of 5 ng CRP mL(-1) and coefficients of variation of <9.1% are obtained.

Aerogels from unaltered bacterial cellulose: application of scCO2 drying for the preparation of shaped, ultra-lightweight cellulosic aerogels.

Bacterial cellulose produced by the gram-negative bacterium Gluconacetobacter xylinum was found to be an excellent native starting material for preparing shaped ultra-lightweight cellulose aerogels. The procedure comprises thorough washing and sterilization of the aquogel, quantitative solvent exchange and subsequent drying with supercritical carbon dioxide at 40 degrees C and 100 bar. The average density of the obtained dry cellulose aerogels is only about 8 mg x cm(-3) which is comparable to the most lightweight silica aerogels and distinctly lower than all values for cellulosic aerogels obtained from plant cellulose so far. SEM, ESEM and nitrogen adsorption experiments at 77 K reveal an open-porous network structure that consists of a comparatively high percentage of large mesopores and smaller macropores.

Magnetic nanoparticle imaging by means of minimum norm estimates from remanence measurements.

In magnetic nanoparticle imaging, magnetic nanoparticles are coated and functionalized to bind to specific targets. After measuring their magnetic relaxation or remanence, their distribution can be determined by means of inverse methods. The reconstruction algorithm presented in this paper includes first a dipole fit using a Levenberg-Marquardt optimizer to determine the reconstruction plane. Secondly, a minimum norm estimate is obtained on a regular grid placed in that plane. Computer simulations involving different parameter sets and conditions show that the used approach allows for the reconstruction of distributed sources, although the reconstructed shapes are distorted by blurring effects. The reconstruction quality depends on the signal-to-noise ratio of the measurements and decreases with larger sensor-source distances and higher grid spacings. In phantom measurements, the magnetic remanence of nanoparticle columns with clinical relevant sizes is determined with two common measurement systems. The reconstructions from these measurements indicate that the approach is applicable for clinical measurements. Our results provide parameter sets for successful application of minimum norm approaches to Magnetic Nanoparticle Imaging.

Development of an immunofiltration-based antigen-detection assay for rapid diagnosis of Ebola virus infection.

Ebola virus (EBOV) has caused outbreaks of severe viral hemorrhagic fever in regions of Central Africa where medical facilities are ill equipped and diagnostic capabilities are limited. To obtain a reliable test that can be implemented easily under these conditions, monoclonal antibodies to the EBOV matrix protein (VP40), which previously had been found to work in a conventional enzyme-linked immunosorbent assay, were used to develop an immunofiltration assay for the detection of EBOV antigen in chemically inactivated clinical specimens. The assay was evaluated by use of defined virus stocks and specimens from experimentally infected animals. Its field application was tested during an outbreak of Ebola hemorrhagic fever in 2003. Although the original goal was to develop an assay that would detect all EBOV species, only the Zaire and Sudan species were detected in practice. The assay represents a first-generation rapid field test for the detection of EBOV antigen that can be performed in 30 min without electrical power or expensive or sensitive equipment.

Immunoassay-based diagnostic point-of-care technology for oral specimen.

We have outlined our progress in developing a novel point-of-care platform to quantify micro-organisms causing dental infections and/or inflammatory markers reflecting an oral disease status. This system is based on a sandwich immunoassay technology known as ABICAP (Antibody Immuno Column for Analytical Processes) using poly-horseradish peroxidase conjugates. This assay enabled us to quantify 500 colony-forming units of Streptococcus sobrinus per milliliter of saliva. The platform allows rapid and convenient performance chairside of such tests by a dentist or dental hygienist within 20 minutes at the dental office.

Magnetic biosensor for the detection of Yersinia pestis.

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.

CRP determination based on a novel magnetic biosensor.

The c-reactive protein (CRP) is a very significant human blood marker for inflammatory processes and is routinely determined for many clinical purposes. The widespread and well established detection method for this approximately 115 kDa hepatic protein is the high-sensitivity ELISA assay (hsCRP-ELISA) in blood serum. New approaches in medical CRP diagnosis (e.g. for CVD, inflammatory bowel disease) require rapid quantification in native matrices. A novel CRP determination method based on magnetic detection is described and tested for human blood serum, saliva and urine. The detection principle is based on two different anti-CRP antibodies (monoclonal, IgG) for CRP trapment and labelling. The linear detection range of this immunosensor ranged from 25 ng/ml to 2.5 microg/ml and is therefore much more sensitive than typical hsCRP-ELISA-assays.