Method: scFv M13 phage display library was created using spleen RNA from a mouse immunized with F. nucleatum. The library was enriched by biopanning against F. nucleatum and individual clones were analyzed by ELISA to identify F. nucleatum specific scFvs. scFvs that inhibit Streptococcus sanguinus interaction with F. nucleatum were identified by coaggregation assays. Selected scFvs were analyzed by Western blotting against F. nucleatum outer membrane proteins, BstOI restriction analysis and DNA sequencing.
Result: The library consisted of 4×108 clones and was enriched by biopanning 6 times. All 292 individual clones tested reacted strongly to F. nucleatum by ELISA. Sixty-two of the 292 clones inhibited F. nucleatum interaction with S. sanguinis. The 62 scFvs were further grouped into 5 categories based on reactivity with F. nucleatum outer membrane proteins by Western blotting. Analysis of 11 representative clones from the 5 groups revealed differences in coaggregation inhibition, recognition of outer membrane proteins, and BstOI restriction pattern. DNA sequencing showed 6 unique scFvs and of them 3 strongly inhibited interaction with 5 Streptococcus species. These scFvs recognize the outer membrane autotransporter protein Fn1893, as determined by mass spectrometry.
Conclusion: A phage display scFv library against F. nucleatum was successfully constructed and from it several scFvs that inhibit coaggregation were identified, opening the door to the identification of F. nucleatum adhesins involved in coaggregation.