Macroporous Monolithic Layers as Efficient 3-D Microarrays for Quantitative Detection of Virus-like Particles (original) (raw)
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Journal of Chromatography A, 2011
A method for flow-through purification of viruses and virus like nano-particles using a combination of binding and size-exclusion chromatography was developed. This technique relies on minimizing the external surface area per unit volume available for virus binding by increasing the mean diameter of the beads used in the column. At the same time the impurity binding capacity of the column is maximized by utilizing beads with multiple functionalities of the optimum size. Purification of different types of viruses and virus-like-particles could be achieved using this technique. Flow-through purification of influenza virus using this technique yielded virus recoveries greater than 70-80% coupled with impurity removal greater than 80%. Finally an approach to optimize and facilitate process development using this technology is presented. Since the impurity binding occurs via a non-specific mechanism and virus recovery is achieved through reduced surface area, the technique is not limited to specific types of viruses and offers the potential as a universal purification tool.
Application of short monolithic columns for improved detection of viruses
Journal of Virological Methods, 2003
Monolithic chromatography media represent a novel generation of stationary phases introduced in the last 10–15 years providing a chromatography matrix with enhanced mass transfer and hydrodynamic properties. These features allow for an efficient and fast separation of especially large biomolecules like e.g., DNA and viruses. In this study, the enrichment of virus RNA on short monolithic columns prior to molecular
Analytical Chemistry, 2008
This research is devoted to the development and optimization of fine purification processes realized on short monolithic columns (CIM disks), using influenza vaccine and viruslike synthetic particles as model objects. The pseudoaffinity mode of liquid chromatography has been used as a tool for dynamic adsorption experiments. Viruslike particles, close to the dimensions of influenza viruses, were developed by means of main antigen of influenza viruses (hemeagglutinin) covalent binding to the outer aminated surface of synthetic latex particles. The natural receptor analogues of sialic acid were used as affinity ligands immobilized on the surface of the CIM disk by different ways to achieve a high adsorption capacity. Also, some other ligands were tested as possible candidates for virus capturing. The affinity binding parameters for influenza A virus were obtained by frontal elution method at optimized chromatographic conditions and immobilization schemes. The experimental data pointed out the possibility of selective isolation of hemeagglutinin from a mixture of vaccine proteins. The results obtained by fast affinity chromatography have shown functional and sterical correspondence viruslike synthetic models to influenza viruses. Additionally, the optimization of chromatographic conditions allowed isolation of influenza virus A while maintaining its virulence. The maximum value of adsorption capacity was registered for a monolithic disk, modified subsequently by chitosan and 2,6sialyllactose and found to be equal to 6.9 × 10 12 virions/ mL support.
The use of monolithic polymeric sorbents to simulate virus-cell interactions
Russian Journal of Applied Chemistry, 2008
A simple virus3cell complementary model system can be obtained using polymer-analogous reactions of the epoxy groups of glycidyl methacrylate3ethylene glycol dimethacrylate monolithic macroporous polymeric support and of the carboxy groups of styrene3methyl methacrylate polymeric nanospheres. The effect of thus designed microenvironment on the affinity binding parameters of virus-mimicking nanoparticles with the functionalized sorbent surface is studied by high-performance monolithic disk affinity chromatography.
Polysaccharide-based chromatographic adsorbents for virus purification and viral clearance
Journal of Pharmaceutical Analysis, 2020
Viruses still represent a significant threat to human and animal health worldwide. In the fight against viral infections, high-purity viral stocks are needed for manufacture of safer vaccines. It is also a priority to ensure the viral safety of biopharmaceuticals such as blood products. Chromatography techniques are widely implemented at both academic and industrial levels in the purification of viral particles, whole viruses and virus-like particles, and to remove viral contaminants from biopharmaceutical products. This paper focuses on polysaccharide adsorbents, particulate resins and membrane adsorbers, used in virus purification/removal chromatography processes. Different chromatographic modes are surveyed, with particular attention on ion exchange and affinity/pseudo-affinity adsorbents among which commercially available agarose-based resins (Sepharose®) and cellulose-based membrane adsorbers (Sartobind®) occupy a dominant position. Mainly built on the development of new ligands coupled to conventional agarose/cellulose matrices, the development perspectives of polysaccharide-based chromatography media in this antiviral area are stressed in a conclusive part.
Applied and environmental microbiology, 1993
Quantification of two plant viruses in suspensions of homoionic Ca-bentonite was conducted by applying to the enzyme-linked immunosorbent assay plates either the virus-clay mixture (direct method) or the supernatant obtained after the clay was allowed to settle (classic method). Both methods showed a similar dependence of free virus content on clay concentration. A higher content at equilibrium was measured for both viruses by the direct method. The advantage of using the direct over the classic method is discussed.
Journal of Separation Science
Hepatitis B virus‐like particles expressed in Escherichia coli were purified using anion exchange adsorbents grafted with polymer poly(oligo(ethylene glycol) methacrylate) in flow‐through chromatography mode. The virus‐like particles were selectively excluded, while the relatively smaller sized host cell proteins were absorbed. The exclusion of virus‐like particles was governed by the accessibility of binding sites (the size of adsorbents and the charge of grafted dextran chains) as well as the architecture (branch‐chain length) of the grafted polymer. The branch‐chain length of grafted polymer was altered by changing the type of monomers used. The larger adsorbent (90 μm) had an approximately twofold increase in the flow‐through recovery, as compared to the smaller adsorbent (30 μm). Generally, polymer‐grafted adsorbents improved the exclusion of the virus‐like particles. Overall, the middle branch‐chain length polymer grafted on larger adsorbent showed optimal performance at 92% f...
Toward the detection of single virus particle in serum
Analytical Biochemistry, 2006
There is a grand challenge for the detection of target molecules at single molecule sensitivity in a bulk body fluid for the early diagnosis of diseases. We report our progress on tackling this challenge via the combination of fluorescence cross-correlation spectroscopy (FCCS) and micro fabricated devices toward highly sensitive detection of the dengue virus. We demonstrate that by using a dengue-specific antibody, we can probe the individual dengue virus in a nanomolar bulk solution by following the specific association of dengue antibody using FCCS. Consequently, we designed and fabricated a microfluidic chamber array structure and were able to compartmentalize the bulk aqueous dengue sample into femtoliter volumes using such a device. More importantly we demonstrate that we can differentiate between the compartments containing the dengue virus and the virus-free compartments. Our experiment suggests that by expanding the throughput using microfluidic devices integrated with FCCS, both of which can be achieved practically, we should be able to detect single virus particle in human body fluids in the near future.
Biotechnology and Bioengineering, 2005
A streptavidin derivitised macroporous monolith was developed to enable single-step capture of chemically biotinylated Moloney Murine Leukaemia Virus (MoMuLV) from crude, unclarified cell culture supernatant. Monoliths were prepared by aqueous cryopolymerisation of acrylamide with N,N″-methylene-bis (acrylamide) and glycidyl methacrylate (Arvidsson et al. [2003] J Chrom A 986:275–290). Streptavidin was immobilised to the epoxy functionalised monoliths. Particulate-containing cell culture supernatant was passed through the monolith without preclarification of the feedstock and adsorption capacities of 2 × 105 cfu/ml of adsorbent were demonstrated (cf. Fractogel streptavidin, at 3.9 × 105 cfu/ml of adsorbent). The specific titre of the recovered fraction was increased by 425-fold; however, recoveries of less than 8% were achieved. Adsorption of nonbiotinylated MoMuLV on the streptavidin-coated monolith was not observed. © 2005 Wiley Periodicals, Inc.
A comparison of labelled antibody methods for the detection of virus antigens in cell monolayers
Journal of Immunological Methods, 1979
A number of labelled antibody methods have been applied to the detection of Semliki Forest virus antigens after replication of the virus in monolayers of host cells in multi-well polystyrene plates. The importance of several reaction variables has been investigated and the sensitivity of the methods compared for different periods of virus replication. Direct assays with radio-labelled antibody (RLA) and indirect assays using peroxidase-antiperoxidase complexes (PAP) were equally sensitive. Direct and indirect assays using enzyme-linked antibodies (ELA) were slightly less sensitive than the direct RLA and PAP methods but were more sensitive than the indirect RLA or fluorescent antibody (FLA) methods. Direct assays using ELA were more rapid and easier to perform than the other assay methods.