Performance of the cobas® HBV RNA automated investigational assay for the detection and quantification of circulating HBV RNA in chronic HBV patients.

BACKGROUND: The amount of HBV RNA in peripheral blood may reflect HBV covalently closed circular DNA (cccDNA) transcriptional activity within infected hepatocytes. Quantification of circulating HBV RNA (cirB-RNA) is thus a promising biomarker for monitoring antiviral treatment. OBJECTIVES: We evaluated the performance of an automated, prototype quantitative HBV RNA assay for use on the Roche cobas® 6800/8800 systems. STUDY DESIGN: The sensitivity, specificity, linearity, and potential interference by HBV DNA of the cobas® HBV RNA assay were assessed using synthetic HBV armored RNA and clinical specimens. RESULTS: cobas® HBV RNA results were linear between 10 and 107 copies/mL in clinical samples of several HBV genotypes, and up to 109 copies/mL with synthetic RNA. Precision and reproducibility were excellent, with standard deviation below 0.15 log10 copies/mL and coefficients of variation below 5% throughout the linear range. The presence of HBV DNA had minimal (<0.3 log10 copies/mL) impact on HBV RNA quantification at DNA:RNA ratios of up to approximately one million. In a panel of 36 untreated patient samples, cirB-RNA concentrations were approximately 200-fold lower than HBV DNA. cirB-RNA was detected in all 13 HBeAg-positive patients (mean 6.0 log10 copies/mL), and in 20 of 23 HBeAg-negative patients (mean of quantifiable samples 2.2 log10 copies/mL). Finally, cirB-RNA was detected in 12 of 20 nucleoside analog-treated patients (mean of quantifiable samples 3.4 log10 copies/mL). CONCLUSIONS: The cobas® 6800/8800 investigational HBV RNA assay is a high throughput, sensitive and inclusive assay to evaluate the clinical relevance of cirB-RNA quantification in patients with chronic hepatitis B.

On the influence of the architecture of poly(ethylene glycol)-based thermoresponsive polymers on cell adhesion.

Thermoresponsive polymer surface coatings are a promising tool for cell culture applications. They allow for a mild way of cell detachment that preserves the activity of membrane proteins-a prerequisite for reliable cell analysis. To enlarge the application range of these coatings to cells with different adhesion properties, we synthesized various novel poly(ethylene glycol)-based thermoresponsive polymers and describe how (i) their chemical structure and (ii) their surface density affect their efficiency. In order to quantify the influence of both factors, the time for cell spreading and rounding efficiency were observed. As a result, efficiency of cell rounding, which is closely correlated to cell detachment, is less affected by both factors than the time needed for cell spreading. This time can effectively be adjusted by the molecular architecture which includes the length of the polymer backbone and the side chains. Based on this work, recommendations are given for future optimization of functionality of thermoresponsive polymer coatings for cell culture applications.

Tuning the mechanical properties of bioreducible multilayer films for improved cell adhesion and transfection activity.

A simple approach to the mechanical modulation of layer-by-layer (LbL) films is through manipulation of the film assembly. Here, we report results based on altering the salt concentration during film assembly and its effect on film rigidity. Based on changes in film rigidity, cell adhesion characteristics and transfection activity were investigated in vitro. LbL films consisting of reducible hyperbranched poly(amide amine) (RHB) have been implemented along with DNA for investigating fibroblast adhesion on [RHB/DNA](n/2) films with varying rigidities. The rigidity was varied by changing the ionic concentration of the deposition solution between 0.01 m NaCl and 1.0 m NaCl. Molecular force probe (MFP) measurements were performed to measure the apparent Young's modulus, E(APP), of the films in situ. Cell adhesion and stress-fiber characteristics were investigated using total internal reflectance microscopy (TIRF-M). The average cell peripheral area, fiber density and average fiber length during 5 days of cell growth on films with either low (below 2.0 MPa) or high (above 2.0 MPa) film elastic modulus were investigated. Transfection studies were performed using gfpDNA and SEAP-DNA to investigate if changes in cell adhesion affect transfection activity. Furthermore, cell proliferation and cytotoxicity studies were used to investigate cellular viability over a week. The results have shown that surface modification of bioreducible LbL films of controlled thickness and roughness promotes cellular adhesion, stress-fiber growth and increased transfection activity without the need for an additional adhesive protein pre-coating of the surface or chemical cross-linking of the film.

Thermoresponsive PEG-based polymer layers: surface characterization with AFM force measurements.

Thermoresponsive polymer-coated surfaces based on poly(2-(2-methoxyethoxy)ethyl methacrylate-co-oligo(ethylene glycol) methacrylate) [P(MEO(2)MA-co-OEGMA)] allow switching between cell attachment and detachment. Here, we investigate the temperature-dependent surface interactions between the polymer coating and a colloidal probe in an aqueous medium by means of atomic force microscopy (AFM) force-distance measurements. The analysis of the adhesion forces from AFM retraction curves identifies two kinds of regimes for the copolymer at temperatures below and above the lower critical solution temperature (LCST). Whereas at 25 degrees C the surface interactions with the polymer in the swollen state are dominated by repulsive forces, at 37 degrees C the surface interactions switch to attractive forces and a stronger adhesion is detected by AFM. Running several heating/cooling cycles repeatedly shows that switching the surface properties provides reproducible adhesion force values. Time-dependent measurements give insight into the switching kinetics, demonstrating that the cell response is coupled to the polymer kinetics but probably limited by the cellular rearrangements.

Single-step electrospinning of bimodal fiber meshes for ease of cellular infiltration.

Bimodal fiber meshes with fiber diameters differing by one order of magnitude, are electrospun in a simple one-step process, using a standard single syringe electrospin setup. The nano- and microfiber meshes combine the benefits of nanofibers (cell adhesion, proliferation) with those of microfibers (open structure, large pore size) and are therefore interesting as scaffolds for cellular infiltration.

Tuning the thickness of polymer brushes grafted from nonlinearly growing multilayer assemblies.

A new versatile method for tuning the thickness of surface-tethered polymer brushes is introduced. It is based on the combination of polyelectrolyte multilayer deposition and surface-initiated atom transfer radical polymerization. To control the thickness of the brushes, the nonlinear growth of certain polyelectrolyte multilayer systems is exploited. The method is demonstrated to work with different polyelectrolytes and different monomers. The relevance for applications is demonstrated by cell adhesion experiments on grafted thermoresponsive polymer layers with varying thickness.

Tuning of thermo-responsive self-assembly monolayers on gold for cell-type-specific control of adhesion.

Self-assembled monolayers (SAMs) on gold containing a thermo-responsive poly( N-isopropylacrylamide)-poly(ethylene glycol)-thiol copolymer were formed. These layers show considerable potential for inducing enzyme-free and gentle detachment of cultivated cells. In an effort to optimize detachment of cells, including strongly adhering ones, two approaches are presented. First, two thermo-responsive copolymers with different poly(ethylene glycol) (PEG) contents of 15 wt % ("P15") and 19 wt % ("P19") were grafted to Au surfaces. Second, mixed monolayers were formed containing P19 and various concentrations of thiol bearing PEG. X-ray photoelectron spectroscopy (XPS) on pure and mixed P19 containing layers confirmed the expected layer compositions. Contact angle measurements showed good functionality of all surfaces prepared. Upon a temperature decrease below the lower critical solution temperature (LCST), the duration until cultivated fibroblasts detached from pure P19 surfaces was half of the one determined on P15. Strongly adherent human osteosarcoma cells could not be detached from pure P19 layers. Through co-adsorption of P19 and thiol-bearing PEG of a molar composition of 1:6, layers were formed that allowed good spreading of osteosarcoma cells above LCST and their efficient detachment below LCST.

Control of cell detachment in a microfluidic device using a thermo-responsive copolymer on a gold substrate.

The control of cell adhesion is crucial in many procedures in cellular biotechnology. A thermo-responsive poly(N-isopropylacrylamide)-poly(ethylene glycol)-thiol (PNIPAAm-PEG-thiol) copolymer was synthesized for the formation of self-assembled monolayers (SAM) that allow the control of adhesion of cells on gold substrates. The contact angle of water on these layers varies between 65 degrees at a temperature of 45 degrees C and 54 degrees at 25 degrees C. This behaviour is consistent with a transition of the polymer chains from an extended and highly hydrated to a collapsed coil-like state. At 37 degrees C, cultivated fibroblasts adhere and spread normally on this surface and detach by reducing the temperature below the lower critical solution temperature (LCST). Layers can repeatedly be used without loss of their functionality. In order to quantify the capability of the copolymer layer to induce cell detachment, defined shear forces are applied to the cells. For this purpose, the laminar flow in a microfluidic device is used. Our approach provides a strategy for the optimization of layer properties that is based on establishing a correlation between a functional parameter and molecular details of the layers.

Double chip protein arrays using recombinant single-chain Fv antibody fragments.

Protein arrays permit the parallel analysis of many different markers in a small sample volume. However, the problem of cross-reactivity limits the degree of multiplexing in parallel sandwich immunoassays (using monoclonal antibodies (mAbs)), meaning antibodies must be prescreened in order to reduce false positives. In contrast, we use a second chip surface for the local application of detection antibodies, thereby efficiently eliminating antibody cross-reactions. Here, we illustrate the potential advantages of using single-chain Fv fragments rather than mAbs as capture and detection molecules with this double chip technology.

Double-chip protein arrays: force-based multiplex sandwich immunoassays with increased specificity.

Protein assays provide direct access to biologically and pharmacologically relevant information. To obtain a maximum of information from the very smallest amounts of complex biological samples, highly multiplexed protein assays are needed. However, at present, cross-reactions of binding reagents restrict the use of such assays to selected cases and severely limit the potential for up-scaling the technology. Here we describe a double-chip format, which can effectively overcome this specificity problem for sandwich immunoassays. This format consists of a capture array and a reference array with fluorescent labeled detection antibodies coupled to the reference array via DNA duplexes. This format allows for the local application of the labeled detection antibodies onto their corresponding specific spots on the capture array. Here we show that this double-chip format allows for the use of cross-reactive antibodies without generating false positive signals, and an assay for the parallel detection of seven different cytokines was set up. Even without further optimization, the dynamic range and the limit of detection for interleukin 8 were found to be comparable to those obtained with other types of multiplexed sandwich immunoassays.