An approach for liposome immobilization using sterically stabilized micelles (SSMs) as a precursor for bio-layer interferometry-based interaction studies

Wallner, Jakob; Lhota, Gabriele; Schosserer, Markus; Vorauer-Uhl, Karola

doi:10.1016/j.colsurfb.2017.03.015

Cited by 14 publications

(16 citation statements)

References 31 publications

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“…Additionally, if the dissociation phase shows less than 5% decrease in signal during the defined dissociation phase, as observed for the lower concentration range of NP protein, a precise determination of the dissociation rate constants ( k d ) is not possible [ 28 , 29 ]. However, it is feasible to calculate an upper limit for the k d (s −1 ) which is given by k d <−ln(0.95)/ t d , where td is the dissociation time in seconds [ 28 , 30 ] Thus, an upper limit for the K D value, calculated by the ratio of kd/ka, resulted in < 0.7 nM, suggesting a strong interaction in the picomolar range. Moreover, for comparison of single batches the observed binding rate ( k obs ) was plotted as a function of the NP concentration and used for the comparison of the single batches.…”

Section: Methodsmentioning

confidence: 99%

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

Klausberger¹,

Duerkop²,

Haslacher³

et al. 2021

EBioMedicine

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Background: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Methods: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. Findings: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. Interpretation: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms.

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Section: Methodsmentioning

confidence: 99%

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

Klausberger¹,

Duerkop²,

Haslacher³

et al. 2021

EBioMedicine

View full text Add to dashboard Cite

show abstract

“…Additionally, if the dissociation phase shows less than 5% decrease in signal during the defined dissociation phase, as observed for the lower concentration range of NP protein, a precise determination of the dissociation rate constants ( k d ) is not possible ( 63, 64 ). However, it is feasible to calculate an upper limit for the k d (s -1 ) which is given by k d <−ln(0.95)/ t d , where td is the dissociation time in seconds ( 63, 65 ) Thus, an upper limit for the K D value, calculated by the ratio of kd/ka, resulted in < 0.7 nM, suggesting a strong interaction in the picomolar range. Moreover, for comparison of single batches the observed binding rate ( k obs ) was plotted as a function of the NP concentration and used for the comparison of the single batches.…”

Section: Methodsmentioning

confidence: 99%

“…Kinetic evaluation of the BLI data is problematic since the dissociation curves are heterogenic. Additionally, if the dissociation phase shows less than 5% decrease in signal during the defined dissociation phase, as observed for the lower concentration range of NP protein, a precise determination of the dissociation rate constants (kd) is not possible (63,64). However, it is feasible to calculate an upper limit for the kd (s -1 ) which is given by kd<−ln(0.95)/td, where td is the dissociation time in seconds (63, 65) Thus, an upper limit for the KD value, calculated by the ratio of kd/ka, resulted in < 0.7 nM, suggesting a strong interaction in the picomolar range.…”

Section: Bio-layer Interferometry (Bli) Measurementsmentioning

confidence: 99%

A comprehensive antigen production and characterization study for easy-to-implement, highly specific and quantitative SARS-CoV-2 antibody assays

Klausberger

Duerkop

Haslacher

et al. 2021

Preprint

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Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection. While first-generation antibody tests have primarily provided qualitative results with low specificity, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Here, we describe two quantitative ELISA antibody tests based on the SARS-CoV-2 spike receptor-binding domain and the nucleocapsid protein. Comparative expression in bacterial, insect, mammalian and plant-based platforms enabled the identification of new antigen designs with superior quality and high suitability as diagnostic reagents. Both tests scored excellently in clinical validations with multi-centric specificity and sensitivity cohorts and showed unprecedented correlation with SARS-CoV-2 neutralization titers. Orthogonal testing increased assay specificity to 99.8%, thereby enabling robust serodiagnosis in low-prevalence settings. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19.

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“…The ability of chemokines to interact with phospholipid liposomes was tested by ELISA and BLI. BLI experiments were performed in an Octet RED384 system (Pall ForteBio, Fremont, CA) essentially as previously described with some modifications [55]. For these experiments, liposomes were prepared in PBS and DSPE-PEGbiot instead of DOPEbiot was used as biotinylated phospholipid to improve the immobilization of the liposomes on the biosensors.…”

Section: Liposome Binding Assays 25mentioning

confidence: 99%

Chemokines as phosphatidylserine-bound ‘find-me’ signals in apoptotic cell clearance

Pontejo

Murphy

2020

Preprint

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Chemokines are positively charged cytokines that attract leukocytes by binding to anionic glycosaminoglycans (GAGs) on endothelial cells for efficient presentation to leukocyte G protein-coupled receptors (GPCRs). The atypical chemokine CXCL16 has been reported to also bind the anionic phospholipid phosphatidylserine (PS), but the biological relevance of this interaction remains poorly understood. Here we demonstrate that PS binding is in fact a widely shared property of chemokine superfamily members that, like GAG binding, induces chemokine oligomerization. PS is an essential phospholipid of the inner leaflet of the healthy cell plasma membrane but it is exposed in apoptotic cells to act as an ‘eat-me’ signal that promotes engulfment of dying cells by phagocytes. We found that chemokines can bind PS in pure form as well as in the context of liposomes and on the surface of apoptotic cells and extracellular vesicles released by apoptotic cells, which are known to act as ‘find-me’ signals that chemoattract phagocytes during apoptotic cell clearance. Importantly, we show that GAGs are severely depleted from the surface of apoptotic cells and that extracellular vesicles extracted from apoptotic mouse thymus bind endogenous thymic chemokines and activate cognate chemokine receptors. Together these results indicate that chemokines tethered to surface-exposed PS may be responsible for the chemotactic and find-me signal activity previously attributed to extracellular vesicles, and that PS may substitute for GAGs as the anionic scaffold that regulates chemokine oligomerization and presentation to GPCRs on the GAG-deficient membranes of apoptotic cells and extracellular vesicles. Here, we present a new mechanism by which extracellular vesicles, currently recognized as essential agents for intercellular communication in homeostasis and disease, can transport signaling cytokines.

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An approach for liposome immobilization using sterically stabilized micelles (SSMs) as a precursor for bio-layer interferometry-based interaction studies

Cited by 14 publications

References 31 publications

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

A comprehensive antigen production and characterization study for easy-to-implement, highly specific and quantitative SARS-CoV-2 antibody assays

Chemokines as phosphatidylserine-bound ‘find-me’ signals in apoptotic cell clearance

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