Low-Volume Label-Free Detection of Molecule-Protein Interactions on Microarrays by Imaging Reflectometric Interferometry

Burger, Jürgen; Rath, Christin; Woehrle, Johannes; Meyer, Philipp; Ammar, Nessim Ben; Kilb, Normann; Brandstëtter, Thomas; Pröll, Florian; Proll, Günther; Urban, G.; Roth, Günter

doi:10.1177/2211068216657512

Cited by 19 publications

(27 citation statements)

References 18 publications

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“…To investigate the mechanism of action of the antibodies inhibiting the RBD-ACE2 interaction we first confirmed the binding of the antibodies to the RBD domain via labelfree single color reflectometry 35 , followed by peptide epitope mapping to determine their binding sites. 15mer peptides covering the entire S protein sequence were immobilized in a microarray and binding of the antibodies to each peptide was evaluated.…”

Section: Most But Not All the Inhibiting Antibodies Occupy The Ace2mentioning

confidence: 99%

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

Bertoglio

Meier

Langreder

et al. 2020

Preprint

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COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein.The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronavirus has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naïve universal antibody gene libraries HAL9/10 anti SARS2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. Furthermore, these antibodies neutralized active SARS-Cov-2 virus infection of VeroE6 cells. All 17 were all able to bind the isolated RBD, four of them with sub-nanomolar EC50. Epitope analysis of the antibodies revealed that six bind at the RBD-ACE2 interface and two on the opposite side of the domain. Universal libraries from healthy donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation. 4/34 Main textIn 2015 Menachery et al. wrote: "Our work suggests a potential risk of SARS-CoV reemergence from viruses currently circulating in bat populations." 1 . Four years later, a novel coronavirus causing a severe pneumonia was discovered and later named SARS-CoV-2. The outbreak started on a sea food market in Wuhan, Hubei province (China) at the end of 2019. The disease was named COVID-19 (coronavirus disease 2019) by the World Health Organization (WHO). Sequencing showed high identity to bat corona viruses (CoV, in particular RaTG13), beta-CoV virus causing human diseases like SARS and MERS and, to a lesser extent, the seasonal CoV hCoV-OC43 and HCov-HKU1 2,3 . The spike (S) protein of SARS-CoV-2, as well as SARS-CoV, binds to the human zinc peptidase angiotensin-converting enzyme 2 (ACE2) which is expressed on lung cells, heart, kidney and intestine cells and acts as receptor for virus entry. S protein consists of the N-terminal S1 subunit, which includes the receptor binding domain (RBD), and the Cterminal S2 subunit which is anchored to the viral membrane and is required for trimerization and fusion of the virus and host membrane 4-6 . The membrane bound host protease TMPRSS2 is responsible for S protein priming by cleavage of specific sites between S1 and S2. In addition to proteolytic activation of the S2' site, conformational changes and viral entry 7-10 .Antibodies against the...

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Section: Most But Not All the Inhibiting Antibodies Occupy The Ace2mentioning

confidence: 99%

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

Bertoglio

Meier

Langreder

et al. 2020

Preprint

Self Cite

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“…PDMS moulding : PDMS flow cells were moulded by using Elastosil RT 607 (Wacker Chemie), as described by Burger et al …”

Section: Methodsmentioning

confidence: 99%

“…Antibody incubation and fluorescence detection : Antibody incubation was performed by using the microfluidic SCORE device reported by Burger et al., which utilised the same flow cell design as that used for protein copying. Prior to measurements, the slides were washed with dH 2 O, in the case of HaloBind, or 0.1× PBS, in the case of Ni‐NTA, and blown dry with N 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Prior to measurements, the slides were washed with dH 2 O, in the case of HaloBind, or 0.1× PBS, in the case of Ni‐NTA, and blown dry with N 2 . Antibodies were diluted to a final concentration of 5 μg mL −1 in PBS with 1 % BSA ( w / v ) . After an initial blocking step, each antibody step was conducted for 10 min at a flow rate of 30 μL min −1 followed by a washing step with PBS at 60 μL min −1 for 5 min.…”

Section: Methodsmentioning

confidence: 99%

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Protein Microarray Copying: Easy on‐Demand Protein Microarray Generation Compatible with Fluorescence and Label‐Free Real‐Time Analysis

et al. 2019

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Protein microarrays are essential to understand complex protein interaction networks. Their production, however, is a challenge and renders this technology unattractive for many laboratories. Recent developments in cell‐free protein microarray generation offer new opportunities, but are still expensive and cumbersome in practice. Herein, we describe a cost‐effective and user‐friendly method for the cell‐free production of protein microarrays. From a polydimethylsiloxane (PDMS) flow cell containing an expressible DNA microarray, proteins of interest are synthesised by cell‐free expression and then immobilised on a capture surface. The resulting protein microarray can be regarded as a “copy” of the DNA microarray. 2 His6‐ and Halo‐tagged fluorescent reference proteins were used to demonstrate the functionality of nickel nitrilotriacetic acid (Ni‐NTA) and Halo‐bind surfaces in this copy system. The described process can be repeated several times on the same DNA microarray. The identity and functionality of the proteins were proven during the copy process by their fluorescence and on the surface through a fluorescent immune assay. Also, single‐colour reflectometry (SCORE) was applied to show that, on such copied arrays, real‐time binding kinetic measurements were possible.

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“…100 μm) can be monitored. Multistep assays were used to demonstrate feasibility [62]. The iRIf and a modified prototype called SCORE were used as a platform for copying microarrays [396,397].…”

Section: Imagingmentioning

confidence: 99%

Critical assessment of relevant methods in the field of biosensors with direct optical detection based on fibers and waveguides using plasmonic, resonance, and interference effects

Gauglitz

2020

Anal Bioanal Chem

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Direct optical detection has proven to be a highly interesting tool in biomolecular interaction analysis to be used in drug discovery, ligand/receptor interactions, environmental analysis, clinical diagnostics, screening of large data volumes in immunology, cancer therapy, or personalized medicine. In this review, the fundamental optical principles and applications are reviewed. Devices are based on concepts such as refractometry, evanescent field, waveguides modes, reflectometry, resonance and/or interference. They are realized in ring resonators; prism couplers; surface plasmon resonance; resonant mirror; Bragg grating; grating couplers; photonic crystals, Mach-Zehnder, Young, Hartman interferometers; backscattering; ellipsometry; or reflectance interferometry. The physical theories of various optical principles have already been reviewed in detail elsewhere and are therefore only cited. This review provides an overall survey on the application of these methods in direct optical biosensing. The "historical" development of the main principles is given to understand the various, and sometimes only slightly modified variations published as "new" methods or the use of a new acronym and commercialization by different companies. Improvement of optics is only one way to increase the quality of biosensors. Additional essential aspects are the surface modification of transducers, immobilization strategies, selection of recognition elements, the influence of non-specific interaction, selectivity, and sensitivity. Furthermore, papers use for reporting minimal amounts of detectable analyte terms such as value of mass, moles, grams, or mol/L which are difficult to compare. Both these essential aspects (i.e., biochemistry and the presentation of LOD values) can be discussed only in brief (but references are provided) in order to prevent the paper from becoming too long. The review will concentrate on a comparison of the optical methods, their application, and the resulting bioanalytical quality.

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Low-Volume Label-Free Detection of Molecule-Protein Interactions on Microarrays by Imaging Reflectometric Interferometry

Cited by 19 publications

References 18 publications

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

Protein Microarray Copying: Easy on‐Demand Protein Microarray Generation Compatible with Fluorescence and Label‐Free Real‐Time Analysis

Critical assessment of relevant methods in the field of biosensors with direct optical detection based on fibers and waveguides using plasmonic, resonance, and interference effects

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