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. 2015 Aug 19:10:5237-47.
doi: 10.2147/IJN.S84800. eCollection 2015.

Real-time analysis of dual-display phage immobilization and autoantibody screening using quartz crystal microbalance with dissipation monitoring

Kaushik Rajaram  1 , Patricia Losada-Pérez  2 , Veronique Vermeeren  1 , Baharak Hosseinkhani  1 , Patrick Wagner  3 , Veerle Somers  1 , Luc Michiels  1
Affiliations

Real-time analysis of dual-display phage immobilization and autoantibody screening using quartz crystal microbalance with dissipation monitoring

Kaushik Rajaram et al. Int J Nanomedicine. .

Abstract

Over the last three decades, phage display technology has been used for the display of target-specific biomarkers, peptides, antibodies, etc. Phage display-based assays are mostly limited to the phage ELISA, which is notorious for its high background signal and laborious methodology. These problems have been recently overcome by designing a dual-display phage with two different end functionalities, namely, streptavidin (STV)-binding protein at one end and a rheumatoid arthritis-specific autoantigenic target at the other end. Using this dual-display phage, a much higher sensitivity in screening specificities of autoantibodies in complex serum sample has been detected compared to single-display phage system on phage ELISA. Herein, we aimed to develop a novel, rapid, and sensitive dual-display phage to detect autoantibodies presence in serum samples using quartz crystal microbalance with dissipation monitoring as a sensing platform. The vertical functionalization of the phage over the STV-modified surfaces resulted in clear frequency and dissipation shifts revealing a well-defined viscoelastic signature. Screening for autoantibodies using antihuman IgG-modified surfaces and the dual-display phage with STV magnetic bead complexes allowed to isolate the target entities from complex mixtures and to achieve a large response as compared to negative control samples. This novel dual-display strategy can be a potential alternative to the time consuming phage ELISA protocols for the qualitative analysis of serum autoantibodies and can be taken as a departure point to ultimately achieve a point of care diagnostic system.

Keywords: dissipation monitoring; dual-display phage; quartz crystal microbalance; rheumatoid arthritis; streptavidin-binding protein; surface plasmon resonance.

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Figures

Figure 1
Figure 1
Δf and ΔD responses upon STV adsorption and subsequent binding of SBP displaying phage SR21 (A and C) and non-SBP displaying CR21 (B and D) to the Au-coated QCM-D sensors. The red arrows indicate the time of addition of the sample. Abbreviations: STV, streptavidin; SBP, streptavidin-binding protein; QCM-D, quartz crystal microbalance with dissipation monitoring.
Figure 2
Figure 2
Kinetic study on the phage immobilization. Notes: (A) ΔD – Δf plots showing adsorption of STV (black solid line) and SBP displaying phage SR21 (red solid line). (B) Estimated thickness of the SBP displaying phage SR21 layer. The red arrows indicate the time of addition of the sample. Abbreviations: STV, streptavidin; SBP, streptavidin-binding protein.
Figure 3
Figure 3
Kinetics of adsorption of STV to Au-coated QCM-D sensors and subsequent binding of SBP displaying phage SR21. Notes: The blue and red solid lines represent the fits to Equation 1: Δf = Δf0 + Ae−1/τ. The black line is experimental data. The red arrows indicate the time of addition of the sample. Abbreviations: STV, streptavidin; QCM-D, quartz crystal microbalance with dissipation monitoring; SBP, streptavidin-binding protein; A, amplitude; τ, time constant.
Figure 4
Figure 4
Characterization of SBP displaying phage binding to the streptavidin surfaces using SPR. Notes: (A) The SBP displaying phage (SR21 and SB) binds to the STV surface of a SA chip and non-SBP displaying phage (CR21, CB) shows no binding pattern. (B) shows the real-time binding curves, and SBP displayers show high stability when flushing with buffer while the negatives revert back to the base line due to the buffer effect. Abbreviations: SBP, streptavidin-binding protein; SPR, surface plasmon resonance; STV, streptavidin.
Figure 5
Figure 5
Characterization of antihuman IgG adsorption and subsequent binding of positive complex (A and C) and negative complex (B and D) to the Au-coated QCM-D sensors. The arrows indicate the time of addition of the sample. Abbreviation: QCM-D, quartz crystal microbalance with dissipation monitoring.
Figure 6
Figure 6
Binding of positive and negative complexes onto the antihuman IgG surface. Notes: ΔD – Δf plots for overtone seven on antihuman IgG adsorption (blue solid line is a linear fit with slope kD–f) and binding of positive complex (A) and negative complex (B) (red solid line is a linear fit with slope kD–f). (C and D) Estimated thickness of the positive complex and the negative complex layers, respectively. The arrows indicate the time of addition of the sample.
Figure 7
Figure 7
Kinetic study on adsorption of IgG to Au-coated QCM-D sensors and subsequent binding of positive complex (A) and negative complex (B) for overtone seven. Notes: The solid lines represent the fits to Equation 1: Δf = Δf0 + Ae−1/τ. The arrows indicate the time of addition of the sample. Abbreviations: QCM-D, quartz crystal microbalance with dissipation monitoring; A, amplitude; τ, time constant.
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