Adaptive virus detection using filament-coupled antibodies

Abstract

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) virionsml, and 40 min was required to detect 10(8) virionsml. In tests of the components of an adaptive strategy, a 30-min virus (3.3 x 10(10) virionsml) incubation time, followed by repositioning the filament-captured virus either within the detecting antibody chamber, (20 microg 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.

Fig. 1

Assay design schematic. Filament-coupled capture antibodies are positioned within a series of five reaction chambers before passing through a fluorescence detector. Filament associated virus is detected fluorescently using diode laser excitation and photomultipliers.

Fig. 2

Optical path of virus detection system. Using a 638-nm laser excitation source, detection of filament fluorescence is possible using the appropriate emission filters. Exposure area on the filament is reduced by an excitation slit.

Fig. 3

Effects of laser power on scan repeatability. Repeated laser scans of virus detection filaments result in a significant signal drop for filaments using full laser power (black bars) but not with reduced laser power (gray bars).

Fig. 4

Representative filament scan showing photomultiplier output as a function of filament position. Laser scanning of the filament detects virus in all three capture antibody regions (anti-M13) with a SNR of approximately 51 ± 4.5. Negative control antibody region (anti-E) is not distinguishable from background. Arrows indicate the location of each immobilized antibody region along the filament.

Fig. 5

Virus incubation times required to detect different virus concentrations. A one-minute virus incubation detected 3.3 × 1010 virions/ml with a SNR of nearly 10. Concentrations below 3.3 × 108 virions/ml were not detectable with a 75-min virus incubation time. Asterisks indicate that a difference in fluorescence signal between capture antibody regions and regions without antibodies is statistically significant (p<0.05).

Fig. 6

Effects of reprocessing on fluorescence. Signal increases as the capture antibody region of the filament is cycled back to the detecting antibody chamber (top) or the virus chamber (bottom) for additional incubation. Arrows indicate regions of immobilized antibodies. The lower sampling rate in the top panel resulted in a smoother curve. Virus concentration was 3.3 × 10¹⁰ virions/ml.

Fig. 7

Comparison of multiple short virus incubations (black bars) to one long virus incubation (gray bars). Virus cumulative incubation times of 1, 5, or 10 min resulted in similar fluorescence for a single filament exposed multiple times or for separate filaments each exposed for the same cumulative time indicated. Virus concentration was 3.3 × 10¹⁰ virions/ml.