Virus detection using filament‐coupled antibodies

Stone, Gregory P.; Mernaugh, Ray; Haselton, Frederick R.

doi:10.1002/bit.20537

Cited by 7 publications

(19 citation statements)

References 16 publications

(14 reference statements)

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“…Our previously described filament-based antibody recognition assay (FARA) employs antibodies immobilized at known locations along a filament to detect specific pathogens 9 , 10. The filament is pulled through a series of small reaction chambers, and pathogens, if present, are captured by filament-bound antibodies.…”

Section: Introductionmentioning

confidence: 99%

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Autonomous Reovirus Strain Classification Using Filament-Coupled Antibodies

et al. 2006

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Abstract-We previously described a filament-based antibody recognition assay (FARA) that generates ELISA-like sandwich structures immobilized on a filament. FARA allows the coupling of antibodies to precise locations along a filament, on-line fluorescence detection of captured pathogen, and feedback-directed filament motion. These properties suggest that this approach might be useful as an automated means to rapidly classify unknown pathogens. In this report, we describe validation of the novel decision tree aspect of this technology using mammalian reovirus. Based on available antibodies, we developed a decision tree algorithm to detect virus with increasing specificity at each level of the tree. Using three strains of reovirus and a bacteriophage control, our system correctly classified the reovirus strains at a concentration of 2 Â 10 12 virions mland M13K07 phage at 3 Â 10 11 virions ml )1. Classification of reovirus strain type 3 Dearing (T3D) required three levels of testing: general reovirus classification in level 1, serotype 3 classification in level 2, and final T3D strain classification in level 3. Strain T3SA + also required three levels of testing before a final classification was returned in level 3. Classification of strain type 1 Lang (T1L) required two levels of testing. M13K07 phage detection required only one level of testing for classification. These results indicate that automated pathogen classification using FARA is feasible. Furthermore, the simplicity of the design could be exploited for development of more complex sub-classification networks with additional levels and branches.

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Section: Introductionmentioning

confidence: 99%

“…FARA was first reported using immobilized anti-M13K07 antibody to detect M13K07 phage 10. This virus and antibody pair provided a well-characterized test system to demonstrate the feasibility of a filament-based, pathogen-detection platform.…”

Section: Introductionmentioning

confidence: 99%

Autonomous Reovirus Strain Classification Using Filament-Coupled Antibodies

et al. 2006

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“…Filaments were immediately fastened to a standard, pre-cleaned microscope slide with tape, and the fluorescence measured using a Genepix 4000B microarray scanner (Axon Instruments) equipped with a 635 nm laser line as previously described (9). The mean fluorescence intensity of ROIs defined along discrete filament spots was measured by Image Pro Plus on grayscale 16-bit images, and plotted using SigmaPlot.…”

Section: Photopatterning Of Antibody On Polyester Filament-based M13kmentioning

confidence: 99%

Light-Guided Surface Engineering for Biomedical Applications

2008

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Free radical species generated through fluorescence photobleaching have been reported to effectively couple a water-soluble species to surfaces containing electron-rich sites (1). In this report, we expand upon this strategy to control the patterned attachment of antibodies and peptides to surfaces for biosensing and tissue engineering applications. In the first application, we compare hydrophobic attachment and photobleaching methods to immobilize FITC-labeled anti-M13K07 bacteriophage antibodies to the SiO 2 layer of a differential capacitive biosensor and to the polyester filament of a feedback-controlled filament array. On both surfaces, antibody attachment and function were superior to the previously employed hydrophobic attachment. Furthermore, a laser scanning confocal microscope could be used for automated, software-guided photoattachment chemistry. In a second application, the cell-adhesion peptide RGDS was site-specifically photocoupled to glass coated with fluorescein-conjugated poly(ethylene glycol). RGDS attachment and bioactivity were characterized by a fibroblast adhesion assay. Cell adhesion was limited to sites of RGDS photocoupling. These examples illustrate that fluorophore-based photopatterning can be achieved by both solution-phase fluorophores or surface-adhered fluorophores. The coupling preserves the bioactivity of the patterned species, is amenable to a variety of surfaces, and is readily accessible to laboratories with fluorescence imaging equipment. The flexibility offered by visible light patterning will likely have many useful applications in bioscreening and tissue engineering where the controlled placement of biomolecules and cells is critical, and should be considered as an alternative to chemical coupling methods.

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“…The monofilament was wound around the ends of a PhastGel sample applicator (Amersham Biosciences) and placed within the concave teeth as described previously. 13 The comb/ filament apparatus was washed in 70% ethanol, rinsed in water, washed in 10% HCl, and rinsed again. The filament was dried and 0.75 μl of capture antibody solution (500 μg/ml) was pipetted onto each tooth of the comb.…”

Section: Antibody Immobilization On Filamentmentioning

confidence: 99%

“…13 We now report the integration of an on-line optical detection method to enable adaptive feedback control of virus detection. Briefly, FARA is a sandwich-based immunoassay in which virus capture antibodies are immobilized on the surface of a monofilament rather than on a polystyrene plate in typical enzyme-linked immunosorbent assay (ELISA).…”

Section: Introductionmentioning

confidence: 99%

Adaptive virus detection using filament-coupled antibodies

2006

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We recently reported the development of a filament-antibody recognition assay (FARA), in which the presence of virions in solution initiates the formation of enzyme-linked immunosorbent assay (ELISA)-like antibody complexes. The unique features of this assay are that processing is achieved by motion of a filament and that, in the presence of a virus, antibody-virus complexes are coupled to the filament at known locations. In this work, we combine the unique features of this assay with a 638-nm laser-based optical detector to enable adaptive control of virus detection. Integration of on-line fluorescence detection yields approximately a five-fold increase in signal-to-noise ratio (SNR) compared to the fluorescence detection method reported previously. A one-minute incubation with an M13K07 test virus is required to detect 10 10 virions/ml, and 40 min was required to detect 10 8 virions/ml. In tests of the components of an adaptive strategy, a 30-min virus (3.3 × 10 10 virions/ ml) incubation time, followed by repositioning the filament-captured virus either within the detecting antibody chamber, (20μg/ml) or within the virus chamber, found an increase in signal roughly proportional to the cumulative residence times in these chambers. Furthermore, cumulative fluorescence signals observed for a filament-captured virus after repeated positioning of the filament within the virus chamber are similar to those observed for a single long incubation time. The unique features of the FARA-like design combined with online optical detection to direct subsequent bioprocessing steps provides new flexibility for developing adaptive molecular recognition assays.

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Virus detection using filament‐coupled antibodies

Cited by 7 publications

References 16 publications

Autonomous Reovirus Strain Classification Using Filament-Coupled Antibodies

Autonomous Reovirus Strain Classification Using Filament-Coupled Antibodies

Light-Guided Surface Engineering for Biomedical Applications

Adaptive virus detection using filament-coupled antibodies

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