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

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.

SUBMITTER: Rajaram K

PROVIDER: S-EPMC4547655 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Rajaram Kaushik K Losada-Pérez Patricia P Vermeeren Veronique V Hosseinkhani Baharak B Wagner Patrick P Somers Veerle V Michiels Luc L

International journal of nanomedicine 20150819

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 autoanti ...[more]

PMID: 26316752

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Project description:BackgroundThe presence of lignin normally affects enzymatic saccharification of lignocellulose detrimentally. However, positive effects of lignin on enzymatic hydrolysis have been recently reported. Enzyme-lignin interactions could be the key to reveal the underlying mechanism of their discrepant behaviors. In this study, to elucidate the positive effects of extractable lignin (EL) on enzymatic hydrolysis of ethanol organosolv-pretreated wood sawdust, two lignin fractions, EL and milled wood lignin (MWL), were isolated sequentially from pretreated substrates. Quartz crystal microbalance with dissipation (QCM-D) was then used to investigate the lignin aggregation effects on enzyme adsorption.ResultsWe found that both EL and MWL had a narrow molecular weight distribution. However, MWL had an obviously higher molecular weight than EL. This indicated that EL and MWL likely represent two distinct lignin fractions from ethanol organosolv-pretreated substrates. HSQC NMR analysis revealed that less β-O-4, β-β, and β-5 linkages and a higher S/G ratio was present in EL, as compared to MWL. QCM-D analysis showed that the enzyme adsorption on lignin was highly relevant to these lignin structural characteristics. An obviously lower maximum enzyme adsorption capacity was observed on EL films (152.63-168.09 ng/cm2) compared to MWL films (196.71-224.73 ng/cm2). Furthermore, enzyme desorption on lignin films was determined. A significantly lower irreversible enzyme adsorption was observed on EL (75.40 ng/cm2) compared to MWL (137.35 ng/cm2). More importantly, two reconstructed lignin films were designed to investigate lignin assembly on enzyme adsorption. The results indicated that the presence of EL reduced irreversible enzyme adsorption on the reconstructed lignin films by 39.2-45.0%.ConclusionsLignin structure determined the interaction between enzyme and lignins. A positive correlation was observed between molecular weight, the content of β-5 linkages, and enzyme adsorption on lignin. EL, which was more depolymerized and less condensed, had the lower enzyme adsorption of the two preparations tested. Additionally, the presence of EL reduced enzyme adsorption on reconstructed lignin films, perhaps through a mechanism involving the blocking of non-productive enzyme binding sites on the MWL. This could be the mechanism for the positive effects of EL on enzymatic hydrolysis.

Dataset Information

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

Publications

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

Similar Datasets

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