Current antibody-based methods used for virus detection are cumbersome, have limited sensitivity due to substantial background staining, and are costly as they often require multiple antibodies and enzyme substrate for detection. In an effort to ameliorate these issues, and reduce the time for virus detection, we synthesized semiconductor CdTe QDs with emission at either 540 nm or 585 nm to be conjugated to monoclonal antibodies to detect RSV-infected cells. These bioconjugated NPs (RSV-NPs) that are specific to RSV F protein were tested for their ability to detect RSV infection of cells both in vitro and in vivo. As expected, no differences in virus detection were observed using either 540 or 585 nm NPs.
As shown in the high resolution transmission electron microscopy (HRTEM) images in , the green emission CdTe NPs had an average size of approximately 3 nm (A) and the orange CdTe NPs of 5 nm (B). Given the reported difficulties conjugating proteins to CdTe NPs (Tan et al 2004
; Gao et al 2005
; McVeigh 2006
), the CdTe NPs were produced with a thioglycolate coating to allow direct anti-RSV F protein monoclonal antibody conjugation via an EDC/Sulfo-NHS conjugation reaction. These bioconju-gated NPs (RSV-NPs) were stable for >1 year at 4 °C, and did not precipitate in the PBS diluent at room temperature or at 4 °C.
To determine the ability of the RSV-NPs to detect RSV infection, in vitro studies were performed using RSV-infected Vero cells. In these studies, we compared RSV detection using conventional antibody staining methods, ie, primary and secondary antibody staining plus enzyme substrate, to that of a single-step detection method using RSV-NPs. Conventional antibody staining methods produced substantial background staining in mock-infected Vero cells (), however did detect RSV plaque formation in the Vero cell lawn (). In contrast, RSV-NPs used in a single-step method produced little background (), and readily detected RSV plaques (). These results show that RSV-NPs can be used to detect RSV in a single-step fashion, and that this method can be used to greatly reduce the amount of time and reagent needed for RSV detection compared to conventional immunostaining procedures (Tripp et al 1999
Figure 2 RSV-NP detection of RSV-infected Vero cells. Vero cells were mock-infected (A, C) or RSV-infected at a MOI = 1 (B, D) and fixed with acetone: methanol (60:40). Cells were immunostained with by conventional methods (A, B) or by RSV-NPs (C, D), and analyzed (more ...)
Extending the in vitro detection findings (), we determined if RSV-NPs could be used to quantitate RSV titers in a single step fashion, thereby shortening the detection time needed to quantitate virus titers by plaque assay (). In these studies, we compared a conventional immunostaining plaque assay which requires 5 or 6 days of in vitro culture post-RSV infection (Tripp et al 1999
) to rapid single-step detection by RSV-NPs. For comparison of the different plaque assay methods, virus titers were assessed at days 2, 3, 5, or 6 pi. The results showed that at days 5 or 6 pi, conventional immunostaining () and the single-step RSV-NP detection method () were similarly sensitive for detecting RSV plaques in the Vero cell lawn. However, RSV-NPs could detect RSV plaques earlier (days 2 or 3 pi) compared with conventional immunostaining which was comparatively ineffective (), indicating that the single-step RSV-NPs procedure was more sensitive at detecting limiting levels of RSV. This result is not unexpected given the multivalent properties of RSV-NPs.
Figure 3 RSV-NP virus plaque assay. RSV was serially diluted ten-fold and the dilutions used to infect Vero cells for determination of virus titers. The viral titer determined from a conventional immunostaining plaque assay (A). were compared with RSV-NPs detection (more ...)
Figure 4 Dynamic range and sensitivity of detection of RSV-NP plaque assay compared with a conventional plaque assay. The viral titer of RSV-infected Vero cells was determined at day 3 pi (A) and day 5 pi (B) using conventional immunostaining plaque assay or single-step (more ...)
To evaluate the efficacy of RSV-NPs to detect RSV infection in vivo, BALB/c were either intranasally infected with 106 pfu RSV or mock-treated, and at day 4 pi, RSV-infected or mock-treated mice were intravenously administered 0.05 ml of RSV-NPs from the stock solution (0.625 mM) by tail vein. At day 5 pi, the lungs from RSV-NP-treated or mock-treated mice were removed, frozen, and prepared for thin sectioning and IHC analysis using conventional methods, i.e. primary and secondary antibody staining, or directly visualized by fluorescence microscopy in the case of RSV-NP treatment. Treatment with RSV-NPs provided clear and rapid detection of RSV-infected lung tissue () with very limited background staining as assessed in lung tissue from mock-treated mice similarly administered RSV-NPs (). Importantly, the magnitude and location of RSV-NPs staining along the epithelial cells of the alveoli in the lungs was similar to that of conventional IHC staining (), and the background staining for RSV-NPs was lower in naïve mice compared to conventional IHC staining (). The selective staining by RSV-NPs of only RSV-infected lung epithelial cells suggests antibody-mediated specific targeting to sites of infection. These results suggest that RSV-NPs may be useful to determine the progression of RSV infection in the lungs, and as a result, aid our understanding of sites of infection, and help to define disease intervention strategies that employ bioconjugated NP targeting strategies.
Figure 5 RSV-NPs detection of RSV-infected lung tissue. Lung tissue sections from RSV-infected (A, C) or naïve, mock-treated BALB/c mice (B, D) were stained by IHC using RSV-NPs (A, B), or by conventional IHC (C, D), and analyzed using an immunofluorescence (more ...)