A non-competitive detection of analytes in the dip-stick format is more desirable than competitive detection due to high sensitivity. In order to examine the limit of analyte detection using the nanophage dip-stick assay, direct detection using collagen at the test (T) line instead of fibronectin-specific monoclonal as outlined in Figure 4.4. Briefly, serial 2-fold dilutions of analyte (fibronectin) starting from 1 µg per assay (50 µl) were mixed with a constant number of Rnano3FnB nanophage particles (1×1011 per assay). Sticks containing 1µg of collagen at the test (T) line and 0.5µg of anti-pVIII antibody at the control (C) line were allowed to stand in the solution for 30 minutes. In this direct assay, phage particles bound to analyte in sample solution will bind the test (T) line, hence the intensity of the signal at that line is expected to positively correlate with the concentration of analyte. This was observed in assay dipsticks (Figure 4.9A); at the highest concentration of fibronectin (1 μg) the signal at the test line was the strongest, whereas the weakest signal was observed the lowest concentration (3.9 ng; Figure 4.9A, stick 9). The control Rnano3 nanophage particles that do not display FnB domain produced signal only at the C line (Figure 4.9A, sticks 11 and 12).
Densitometric analysis (Figure 4.9B) of signal at the test line indicated a second order polynomial dependence between the pixel density of the signal and analyte concentration over a range of concentrations between 62.4 ng and 500 ng. This range is much broader than the physiological concentration of fibronectin in the human serum which is 259-400 ng/μl. Assay was found to be highly sensitive in general, as it could detect fibronectin concentrations as low as 3.9 ng.
The result of these dipstick immunoassays confirmed that nanophage particles displaying high-affinity analyte-binding antibodies, proteins or peptides on at the pIII end of the particles can be used to detect the cognate analyte in the solution.
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B
Figure 4.9 Non-competitive detection of analyte in dipstick format using nanophage particles.A) Sticks contained 1 µg of collagen at the test line (T) and 0.5 µg anti pVIII antibody at the control line (C). Sample solutions contained serial two fold dilutions of the analyte (fibronectin). Strips 1-11 were dipped into a series of solutions containing a serial two-fold dilutions fibronectin concentration starting with 1000 ng (in 50 µl as indicated above the strips. The control strip 12 was dipped into the solution containing Rnano3 nanophage (that does not display FnB domain) and 1000 ng of fibronectin in 50 µl. B) Plot of the signal intensity at the test (T) line vs. fibronectin concentration in the test solution. The T line signal intensity was determined by densitometry. The X-axis indicates fibronectin amount in the total volume of analyst solution (50 μl), whereas Y-axis indicates band intensity.
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Chapter 5
Application of Nanophage in Vaccine Design
5.1 Introduction
Filamentous phage have been reported to elicit strong antibody response, even when applied in low doses and in the absence of adjuvant (van Houten et al., 2010). This is possibly because of high copy-number and highly repetitive arrangement of the major coat protein in the filamentous virion and particulate nature of the phage, allowing phage to serve both as an antigen carrier and adjuvant. Furthermore, it has been shown that immune response is better focused on the antigens conjugated to filamentous phage, relative to the same antigens conjugated to the standard antigen carriers (van Houten et al., 2006). However, despite these advantages over the standard antigen carriers, application of bacteriophage as antigen carriers in human vaccination is just at the proof of concept stage, mainly because of the consumer and regulatory concerns stemming from the fact that Ff bacteriophage are viruses and are capable of replicating within E. coli that is part of the gut microbial community (Dabrowska et al., 2005). The recombinant phage used in vaccination are genetically modified organisms; their genomes in most cases contain antibiotic resistance markers that can be transferred to commensal and pathogenic bacteria within the gut; they have also been shown to be taken up un-specifically by eukaryotic cells, resulting in delivery of viral DNA into the nucleus and integration into the chromosome (Larocca et al., 1999). In this chapter, I report that nanophage can be used as vaccine carriers, removing some of the concerns mentioned above, linked to the use of the full-length phage as vaccine carriers.
Nanophage are nanoparticles that are not capable of replicating without a helper phage, yet they retain their ability to act as display scaffold in the same fashion as full-length phage particles (this study, Chapter 4). They have an additional advantage of increased heat and detergent resistance over the full-length phage and are non-toxic and non- infectious (this study, Chapter 3, Section 3.3). Because of these advantages, nanophage could be a viable option for use outside laboratory containment, as vaccine carriers. To
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examine whether immunogenic properties of the nanophage are similar to those of the full-length phage, the functionalised nanophage displaying FnB domain as fusion to pIII were purified and tested for induction of antibody response, in comparison to equivalently functionalised full-length phage. Fibronectin binding domain (antigen) is derived from the Serum opacity factor, shown previously to be a virulence factor in
Streptococcus pyogenes (Group A Streptococci or GAS). The FnB domain mediates tissue invasion by these pathogenic bacteria through interaction with fibronectin from the extracellular matrix (Courtney et al., 1999). The FnB domain has been shown to generate protective immune response against S. pyogenes challenge in a mouse model, when administered in conjunction with standard antigen carriers (Schulze et al., 2001).
5.2 Large-scale Purification of Nanophage and Full-length Phage Particles for