Filamentous phage have found extensive applications in the in vitro diagnostics in biomedical industry (Chapter 1, Section 1.7). A relatively new application of filamentous phage to detect the small molecules in a non-competitive anti- immunocomplex assay has been published (Gonzalez-Techera et al., 2007; González- Techera et al., 2007; Kim et al., 2009). The phage-displayed peptides were found to be superior to monoclonal antibodies derived by standard immunization protocols in detecting the changes in immune complexes upon binding of small molecules that cannot be detected by standard sandwich assays, because they cannot bind two different antibodies at the same time. Currently, availability of commercial phage display peptide libraries offers flexibility and cheap access to the technique in comparison to the production of specific monoclonal antibodies. This assay, named Phage Anti-immune complex Assay (PHAIA), was adapted to a simple dipstick format for onsite detection of the analytes. This application indicated the potential use of functionalized full-length phage particles in lateral flow devices such as dipstick for the affinity detection of specific ligands. The dip-stick assays involving the intact phage virion, however, are not used as yet outside the laboratory containment.
In this study I demonstrate the use of the nanophage particles carrying FnB domain as pIII fusion in a setup that corresponds to simple lateral flow dipstick device for the detection of dissolved fibronectin in sample solution. The assay was carried out using protocol listed in Chapter 2 Section 2.7, using a standard dipstick device that contains immobilized mouse monoclonal (mmAb) IgG1 antibody against human fibronectin sprayed at the test (T) line and anti-pVIII antibody sprayed at control (C) line. Phage particles having FnB domain as pIII fusion were expected to bind the anti-fibronectin antibody at the test line only when fibronectin is present in the test solution. However, results indicated a non-specific binding of both full-length and nanophage particles
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carrying FnB domain, in the absence of added analyte (fibronectin) to test line containing fibronectin binding antibody (Chapter 4, Figure 4.5). Since control phage without FnB domain was not binding to the fibronectin antibody, this was indicative of binding due to the displayed FnB and not due to non-specific binding of the phage particles. It is known from literature that fibronectin binding protein Sfb1 from
Streptococcus pyogenes exhibits a non-immune interaction with F(ab)ˈ2 region of mouse IgGs, which is mediated by its fibronectin binding domain (Medina et al., 2000); since FnB domain is conserved in different fibronectin binding proteins it was concluded that FnB domain from serum opacity factor of S. pyogenes displayed on the surface of full-length and nanophage particles in this study was exhibiting a similar antigen-independent interaction. In order to verify this possibility binding of functionalized phage particles with three unrelated mouse monoclonal IgG antibodies was tested in dipsticks. Phage particles were not found to cross-react with any of them (data not shown). This eliminated the possibility of non-specific binding of FnB to the constant portion of antibodies; instead, it pointed to detection of the small amount of serum fibronectin that was co-purified with fibronectin-specific antibodies. This suggests an extremely high sensitivity of detection of analyte using the nanophage particles.
In order to overcome this problem, collagen was used as capture reagent on the test line to capture phage FnB-fibronectin complex. Collagen is known to bind fibronectin with high affinity and is used in sepharose columns to remove fibronectin from the serum samples (Balian et al., 1979; Engvall et al., 1978; Hayman and Ruoslahti, 1979; Ruoslahti et al., 1978). Collagen exhibited specific binding of phage to test line in presence of fibronectin only. Both full-length and nanophage particles exhibited specific binding to fibronectin via FnB domain that was displayed as pIII fusion. Control phage (Rnano3) did not bind to the collagen test line in presence or absence of fibronectin as expected. This indicated the potential for the use of functionalized nanophage particles as probes to detect soluble analyte in any sample solution.
Quantity of the analyte can be a critical parameter in dipstick assays; nanophage particles were also tested for the quantitative detection of fibronectin in sample solution in competitive as well as non-competitive assays. Difference in signal at the test line was found to be consistent with the difference in concentration of analyte in
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the test solutions as indicated by the band density analysis. Both competitive and non- competitive assay showed quantitative correlation in the physiological range of fibronectin in human plasma that allows quantitation of fibronectin in unknown samples within the range of 7.81 ng – 500 ng. (Figure 4.8 and 4.9)
In conclusion, nanophage particles can be easily applied as detector probes in lateral flow dipstick devices for the detection of soluble ligands, offering full advantages associated with filamentous phage structure especially phage display. However both full-length and nanophage particles were not labeled to produce a visible signal, therefore western blotting was carried out to visualize the bound phage. It would be desirable to get a visible signal for onsite detection of the analytes that can be achieved by labeling the phage particles with visible dyes. Attempts were made to do labeling with (Uniblue A) but were unsuccessful due to high hydrophobicity of the dye (data not shown). In order to reduce the processing time of the dipsticks to visualize the bound phage at test line phage were labelled with FITC and detected by phosphorimager. It is already established from literature that phage clones labelled with FITC at major coat protein pVIII, retain the sensitivity and binding ability of the peptides expressed on pIII to the target ligand (Jaye et al., 2004). It was indeed observed as both nanophage and full-length phage demonstrated specific binding to the test line in presence of fibronectin in the test solution. Moreover, nanophage have an additional advantage of being non-infectious particles in comparison to full-length phage therefore they are much more likely to be approved by the regulatory authorities and consumers.