Construction of helper plasmid-mediated dual-display phage for autoantibody screening in serum.
M13 filamentous bacteriophage has been used in displaying disease-specific antibodies, biomarkers, and peptides. One of the major drawbacks of using phage in diagnostic assays is the aspecific adsorption of proteins leading to a high background signal and decreasing sensitivity. To deal with this, we developed a genetically pure, exchangeable dual-display phage system in which biomarkers and streptavidin-binding protein (SBP) are displayed at opposite ends of the phage. This approach allows for sample purification, using streptavidin-coated magnetic beads resulting in a higher sensitivity of signal detection assays. Our dual-display cassette system approach also allows for easy exchange of both the anchor protein (SBP) and the displayed biomarker. The presented principle is applied for the detection of antibody reactivity against UH-RA.21 which is a good candidate biomarker for rheumatoid arthritis (RA). The applicability of dual-display phage preparation using a helper plasmid system is demonstrated, and its increased sensitivity in phage ELISA assays using patient serum samples is shown.
Biomedical Research Institute, Hasselt University, Martelarenlaan 42, 3500, Hasselt, Belgium, rkaushikmbt@gmail.com.
Kaushik
K
Rajaram
Veronique
V
Vermeeren
Klaartje
K
Somers
Veerle
V
Somers
Luc
L
Michiels
D001323:Autoantibodies / Q000097:blood*; D061505:Cell Surface Display Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D017912:Inovirus / Q000235:genetics*; D008403:Mass Screening / Q000379:methods*; D010957:Plasmids; D012680:Sensitivity and Specificity; D044967:Serum / Q000276:immunology*
D001323:Autoantibodies