Authors

Discussion

BACKGROUND

Acanthamoeba is a free-living opportunistic protozoan that can cause serious human infections, the most common being  Acanthamoeba Keratitis (AK).  Early diagnosis and fast treatment are required for good prognosis, however the lack of good tools for rapid identification oftecn cause significant delay in treatment.Current methods of detection involve culture and microscopy. These methods are time consuming, laborious, and open to error. The development of a rapid, simple detection method for Acanthamoeba is thus important.

Phage display is a technology that allows the presentation of peptides and proteins including antibody fragments on the surface of filamentous bacteriophage. The genes encoding the variable domains of antibodies (scFv) and a linker are fused to the g3p gene in the genome of the filamentous phage. In   our system the scFv is displayed as a fusion to g3p (pIII) protein at the tip of the phage.

The aim of this study is to Isolate antibody fragments that can be used to detect Acanthamoeba from clinical samples. It is hoped that these antibodies provide the reagents to establish a specific and rapid detection assay for Acanthamoeba.

METHODS

Sequence analysis of selected clones: Nine clones identified from earlier experiments were cultured in 2xTY broth containing 100 µg/ml ampicillin and 4 %  glucose and incubated overnight at 37°C. The diversity of these clones was determined by PCR screening . Recombinant clones were screened   by amplifying the scoff insert using primers LMB (CAGGAAACAGCTATGAC) and fd-SKEQ1 (GAATTTTCTGTATGAGG).  and the products sequenced

Screen of clones by soluble ELISA: The same clones that sequenced   were used to produce  soluble antibody fragments, briefly, each colony was grown at optimal condition until  and then induced by adding 1 mM Isopropyl-ß-D-thiogalactopyranoside (IPTG) and further incubated for overnight  at 30C and 250 rpm. Cultures were centrifuged and supernatants (soluble fractions) were used in ELISA assay. Binding of soluble scFvs with to Acanthamoeba was detected with the HPR-peroxidase and mouse monoclonal antibody 9E10.

RESULTS

All sequenced clones were used to identify framework and complementarity-determining regions (CDRs) in the VH chain, and compare their amino acids sequence   to the germ line using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). Nucleotide sequence alignment and homology searches were performed with Clustal Omega software. Although there were some differences in the CDRs amino acids sequence, these   clones were 95% identical. ELISA results indicated that the nine selected clones have produced soluble fragments as they showed a high absorbance reading in comparison with control.

CONCLUSION

From our initial work we have identified 9 clones that show significant binding to Acanthamoeba using ELISA, flow cytometry and fluorescent microscopy.

We believe that these novel reagents are suitable candidates as potential tools for Acanthamoeba diagnosis due to cost effectiveness and ease of production. Such reagents may also have potential for use in targeting therapeutic drugs.