Aptamers and Phage as Tools for the Development of Novel and Sensitive Point-of-Care Diagnostics

Immunochromatographic lateral flow assays (LFAs) represent a well-established point-of-care diagnostic analytical method for the primary testing of diverse samples. The sensitivity of conventional LFAs that use the standard colored nanoparticles as reporters lags behind the more elaborate laboratory techniques, such as plaque counting, PCR or ELISA. To address this issue, we have introduced an innovative approach that utilizes functionalized M13 bacteriophage nanoparticles as reporters in the lateral flow diagnostic system. M13 phage offers a multitude of binding sites on its major coat protein pVIII for reporter enzymes, and for affinity agents specific for the target of interest. Furthermore we employed SAM-AviTag phage, derivatives of phage M13 in which the N-terminus of the tail protein pIII is replaced by the enzymatically-biotinylatable AviTag peptide (GLNDIFEAQKIEWHE). The lysine residue (K) in the AviTag is a substrate for biotinylation by the E. coli biotin ligase (birA) enzyme. Using streptavidin or Neutravidin, any biotinylated affinity agent can then easily be linked to these enzymatically-biotinylated phage particles. Viral nanoparticles were functionalized with target-specific antibodies and multiple copies of an enzymatic reporter (horseradish peroxidase). These particles were successfully integrated into a lateral-flow immunochromatographic assay detecting MS2 virus, a model for viral pathogens. The limit of detection of the assay was 10^4 MS2/mL, 1000-fold more sensitive than the conventional gold nanoparticle lateral flow assays, and results could easily be evaluated, even without advanced lab instruments.

Aptamers are short, library-selected nucleic acid molecules that can recognize and bind to pre-selected targets with high affinity and selectivity. They have been used as recognition elements in a variety of applications. We screened for DNA aptamers specific to the murine anti-lysozyme antibody, HyHEL-5. During the validation process, however, the originally selected aptamers did not show successful binding. As an alternative, literature DNA aptamers were employed to construct phage displaying aptamers and HRP, which were used as LFA reporters. We describe the first modification of bacteriophage particles with aptamers as bio-recognition elements, and demonstrate their use in ultrasensitive lateral flow assays detecting IgE and PBP2a (Penicillin binding protein) of Staphylococcus aureus.