Detection of parasitic protozoa using peptide and antibody phage display technologies.
Acanthamoeba is an opportunistic parasitic protozoa that causes a variety of disease in humans. These include a severe keratitis, a disseminating skin infection and granulomatous amoebic encephalitis. Although Acanthamoeba infections are rare Acanthamoeba keratitis (AK) is the most commonly observed infection with an approximate annual incidence of 1–2 cases per million people. Acanthamoeba keratitis is a serious eye infection that often result in permanent loss of visual acuity or blindness if not diagnosed quickly and managed effectively. Treatment of this infection is difficult as there are currently few drugs available for use. Early symptoms of AK are similar to those seen with fungal or viral infections thus accurate identification of this organism is vital. Clinical diagnostic options currently available include PCR/sequencing, culture and confocal microscopy. PCR is the most useful approach but this is only performed in specialist laboratories (4 centres in the UK). It has been suggested that corneal confocal microscopy would be the most clinically useful diagnostic tool as this would allow treatment to occur in a timely fashion. However, distinguishing host cells (particularly lymphocytes) from Acanthamoeba can be difficult thus limiting its value as a stand-alone tool for diagnosing AK. This can be overcome by using Acanthamoeba specific fluorescently labelled peptides for detection using confocal microscopy
With this in mind, the aim of this project was focused on generating novel fluorescent peptides that can be used for identification of Acanthamoeba in the clinical environment. For this work bacteriophage antibody (peptide) display technology was used as a source of peptides. This library expresses human antibody viable heavy domains as a construct with minor phage coat protein (pIII) on the surface of bacteriophage. Using a modified panning method, bacteriophage clones from a library bacteriophage clones that bound to Acanthamoeba via the heavy chain peptides were identified using enzyme-linked immunosorbent assays (ELISA). From this screen, 23 bacteriophage clones were identified that showed binding. Further analysis of these clones was performed including binding to the two life cycles forms seen during infection (cysts and trophozoites); from the 23 clones, nine were selected for further study.
The nine selected phage clones were induced to produce soluble antibody fragments (peptides) constructed with a myc Tag for detection. Soluble fragment production by the clones was assayed using ELISA and all nine clones produced peptides. To purify theses antibody fragments for further analysis (size and amino-acid sequence) anti-c-myc Tag (9E10) Affinity GelChromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. This was not successful thus an alternative approach for accessing the peptides was used. The DNA sequences of