Rapid Detection of Hendra Virus Using Magnetic Particles and Quantum Dots (original) (raw)
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Journal of Virological Methods, 2007
Quantum dots (QDots) are fluorescent semiconductor nanocrystals with a narrow emission spectrum, high quantum yield, and excellent photostability. These unique properties of QDots have been utilized to develop a fluorescent binding assay using biotinylated human T cell leukemia virus type 1 (biot-HTLV-1) conjugated with streptavidin-coated Qdots that enabled both qualitative and quantitative analyses of viral binding. The specificity and linearity of the assay was demonstrated utilizing T cells, the primary HTLV-1-susceptible cell population. Furthermore, differential binding of HTLV-1 was analyzed in additional cell types of clinical relevance including primary CD4 + and CD8 + T cells, dendritic cells (DCs), monocytes, bone marrow progenitor cells, and epithelial cells. DCs exhibited maximum binding affinity when compared to other examined cell types except the Jurkat and SUP-T1 T cell lines. Finally, blocking antibodies directed against a putative HTLV-1 receptor on DCs; DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin), were utilized to study the inhibition of HTLV-1 binding to target cells. Overall, these results demonstrated that this novel high throughput assay can be utilized to study the binding of a biotinylated virus and has implications for screening of viral binding inhibitors as well as host membrane proteins that may serve as receptors for viral entry.
Henipavirus microsphere immuno-assays for detection of antibodies against Hendra virus
Journal of Virological Methods, 2014
Hendra and Nipah viruses (HeV and NiV) are closely related zoonotic pathogens of the Paramyxoviridae family. Both viruses belong to the Henipavirus genus and cause fatal disease in animals and humans, though only HeV is endemic in Australia. In general and due to the acute nature of the disease, agent detection by PCR and virus isolation are the primary tools for diagnostic investigations. Assays for the detection of antibodies against HeV are fit more readily for the purpose of surveillance testing in disease epidemiology and to meet certification requirements in the international movement of horses. The first generation indirect ELISA has been affected by non-specific reactions which must be resolved using virus neutralisation serology conducted at laboratory bio-safety level 4 containment (PC4). Recent developments have enabled improvements in the available serology assays. The production of an expressed recombinant truncated HeV G protein has been utilised in ELISA and in Luminex-based multiplexed microsphere assays. In the latter format, two Luminex assays have been developed for use in henipavirus serology: a binding assay (designed for antibody detection and differentiation) and a blocking assay (designed as a surrogate for virus neutralisation). Equine and canine field sera were used to evaluate the two Luminex assays relative to ELISA and virus neutralisation serology. Results showed that Luminex assays can be effective as rapid, sensitive and specific tests for the detection of HeV antibody in horse and dog sera. The tests do not require PC4 containment and are appropriate for high throughput applications as might be required for disease investigations and other epidemiological surveillance. Also, the results show that the Luminex assays detect effectively HeV vaccine-induced antibodies.
Blue Fluorescent Antibodies as Reporters of Steric Accessibility in Virus Conjugates
Bioconjugate Chemistry, 2003
Nonenveloped viruses provide the chemist with large, preassembled polyvalent protein scaffolds for modification. These structures are typically porous to small molecules but not to large ones. The solution-phase structures and reactivities of such assemblies may be substantially different than indicated by X-ray crystal structures. Here, the attachment of organic compounds to either the inside or outside surface of the cowpea mosaic virus (CPMV) coat protein was verified with an indicating antibody-antigen interaction. Antibody binding was subsequently blocked by the installation of poly-(ethylene glycol) chains. These results typify the type of site-specific control that is available with CPMV and related virus building blocks.
Colloid & Polymer Science, 1990
A fluorescence detection system was developed for the analytical ultracentrifuge Spinco model E. Fluorescence is excited by a laser beam which is focussed into the cell and illuminates an area with a dimension of 60 ~tm in radial direction. For scanning the laser beam is moved in radial direction. After passing the cell, the laser beam is quenched by a carbon light trap and a set of optical filters. Fluorescence emission intensity is monitored by a photomultiplier located behind the light trap and the set of filters. The sensitivity of the detection system was tested by applying it to the sedimentation analysis of proteins and nucleic acids. Bovine serum albumin (BSA) was covalently labelled with the fluorescence-dye fluorescein-isothiocyanate (FITC), and its sedimentation coefficient could be determined even if BSA was analyzed in a concentration as low as 10-10 M. Nucleic acids were labelled non-covalently by the intercalating dye ethidium bromide. Only 8 ng RNA were needed for the determination of the sedimentation coefficient. The particular advantages of the fluorescence detection system were exploited for the establishment of a new method for quantitative virus detection. To tobacco mosaic virus (TMV) a monoclonal anti-TMV antibody from mouse was bound, and to this a second, anti-mouse antibody that carried the fluorescence-label HTC was attached. Either by UV-irradiation or by incubation with glutaraldehyde, the first antibody was covalently crosslinked to TMV, and the second antibody to the first. In CsC1 density centrifugation with fluorescence detection as little as 3.2 ng virus/80 IA or 6 x 10 s virus particles/ml were recorded in a well expressed band at the corresponding buoyant density. Tenfold lower concentration would result still in a significant band. The sensitivity compares well with those of the most advanced techniques from immunology. Due to the specific labelling of viruses by antibodies it will be possible to carry out quantitative physical characterization of virus containing samples without purifying the virus. Future applications of the fluorescence detection system and of the virus detection technique are discussed.
Specific gold-labelling of antibodies bound to plant viruses in mixed suspensions
Netherlands Journal of Plant Pathology, 1985
Protein A-gold complexes with gold particle diameters of 7 and 16 nm were prepared and could be stored at 4 ~ for at least 5 months without loosing activity. The complexes were used to detect antibodies bound to two plant viruses in mixed suspensions. Depending on the antibodies used, each virus could be labelled specifically with protein A-gold complexes with a gold particle diameter of either 7 nm or 16 nm. A double-labelling technique was developed by which the viruses in suspension could be labelled specifically with protein A-gold complexes with gold particle diameters of either of the two sizes mentioned. Using this technique it was possible to distinguish and identify two viruses with a similar spherical appearance in the electron microscope in mixed preparations.
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.
BioMed Research International, 2020
Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10−7 and 1.35 × 10−8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocali...
Surface capturing of virion-antibody complexes: Kinetic study
Materialwissenschaft und Werkstofftechnik, 2013
The presence of various components in complex biological samples which interact with inhomogeneous sensor surface, results in stretched exponential behaviour of the sensor response. This general concept was demonstrated by surface plasmon resonance (SPR) measurements of virusantibody complexes from crude plant homogenates on a gold surface, covered with protein A. The development of the viral infection in the plant Nicotiana tabacum was studied by SPR and ELISA (enzyme-linked immunosorbent assay) as an indicative approach; a good agreement between both methods was shown. The correlation between virus concentration and the parameters of stretched exponential law was demonstrated. These results indicate that the parameters have hidden intrinsic properties of the underlying interfacial reactions and give a good basis for a more specific microscopic model.