Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Probing environmental and tectonic changes underneath Mexico City with the urban seismic field

The sediments underneath Mexico City have unique mechanical properties that give rise to strong site effects. We investigated temporal changes in the seismic velocity at strong-motion and broadband seismic stations throughout Mexico City, including sites with different geologic characteristics ranging from city center locations situated on lacustrine clay to hillside locations on volcanic bedrock. We used autocorrelations of urban seismic noise, enhanced by waveform clustering, to extract subtle seismic velocity changes by coda wave interferometry. We observed and modeled seasonal, co- and post-seismic changes, as well as a long-term linear trend in seismic velocity. Seasonal variations can be explained by self-consistent models of thermoelastic and poroelastic changes in the subsurface shear wave velocity. Overall, sites on lacustrine clay-rich sediments appear to be more sensitive to seasonal surface temperature changes, whereas sites on alluvial and volcaniclastic sediments and on bedrock are sensitive to precipitation. The 2017 Mw 7.1 Puebla and 2020 Mw 7.4 Oaxaca earthquakes both caused a clear drop in seismic velocity, followed by a time-logarithmic recovery that may still be ongoing for the 2017 event at several sites or that may remain incomplete. The slope of the linear trend in seismic velocity is correlated with the downward vertical displacement of the ground measured by interferometric synthetic aperture radar, suggesting a causative relationship and supporting earlier studies on changes in the resonance frequency of sites in the Mexico City basin due to groundwater extraction. Our findings show how sensitively shallow seismic velocity and, in consequence, site effects react to environmental, tectonic and anthropogenic processes. They also demonstrate that urban strong-motion stations provide useful data for coda wave monitoring given sufficiently high-amplitude urban seismic noise.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

FUNCTIONALLY IMPAIRED ANTIBODY RESPONSE TO BNT162B2 BOOSTER VACCINATION IN CVID IgG RESPONDERS.

BACKGROUND: While previous studies described the production of IgG-antibodies in a subgroup of CVID-patients following mRNA-vaccinations with bnt162b2 SARS-CoV2 (CVID responders), the functionality of these antibodies in terms of avidity as measured by the dissociation rate constant (kdis) and the antibody response to booster immunization has not been studied. OBJECTIVE: In CVID responders and healthy individuals the avidity of anti-SARS-CoV-2 serum-antibodies and their neutralization capacity as measured by surrogate virus neutralizing antibodies were analyzed in addition to IgG-, IgM- and IgA-antibody levels and the response of circulating follicular T-helper cells after a third vaccination with BNT162b2 SARS-CoV2 mRNA-vaccine. METHODS: Binding IgG, IgA and IgM serum levels were analyzed by ELISA in CVID-patients responding to the primary vaccination (CVID responders, n=10) and healthy controls (n=41). The binding-avidity of anti-spike antibodies was investigated using biolayer interferometry in combination with biotin-labelled receptor-binding-domain (RBD) of SARS-CoV2 spike-protein and streptavidin-labelled sensors. Antigen-specific recall T-cell responses were assessed by measuring activation-induced markers by flow cytometry. RESULTS: After the third vaccination with BNT162b2 IgG-, IgM and IgA-antibody levels, sVNT levels and antibody avidity were lower in CVID responders as compared to healthy. In contrast αSpike-avidity was comparable in CVID responders and healthy individuals following primary vaccination. Follicular T-helper cell response to booster vaccination in CVID-responders was significantly reduced when compared to healthy individuals. CONCLUSION: Impaired affinity-maturation during booster-response provides new insight into CVID pathophysiology.

Surface Subsidence Characteristics and Causes in Beijing (China) before and after COVID-19 by Sentinel-1A TS-InSAR

Surface subsidence is a serious threat to human life, buildings and traffic in Beijing. Surface subsidence is closely related to human activities, and human activities in Beijing area showed a decreasing trend during the Corona Virus Disease 2019 (COVID-19). To study surface subsidence in Beijing before and after the COVID-19 outbreak and its causes, a total of 51 Sentinel-1A SAR images covering Beijing from January 2018 to April 2022 were selected to derive subsidence information by Time Series Interferometry Synthetic Aperture Radar (TS-InSAR). The results of surface subsidence in Beijing demonstrate that Changping, Chaoyang, Tongzhou and Daxing Districts exhibited the most serious subsidence phenomenon before the COVID-19 outbreak. The four main subsidence areas form an anti-Beijing Bay that surrounds other important urban areas. The maximum subsidence rate reached −57.0 mm/year. After the COVID-19 outbreak, the main subsidence area was separated into three giant subsidence funnels and several small subsidence funnels. During this period, the maximum subsidence rate was reduced to −43.0 mm/year. Human activity decrease with the COVID-19 outbreak. This study effectively analysed the influence of natural factors on surface subsidence after excluding most of the human factors. The following conclusions are obtained from the analysis: (1) Groundwater level changes, Beijing's geological structure and infrastructure construction are the main reasons for surface subsidence in Beijing. (2) Seasonal changes in rainfall and temperature indirectly affect groundwater level changes, thereby affecting surface subsidence in the area. (3) The COVID-19 outbreak in early 2020 reduced the payload of Beijing's transportation facilities. It also slowed down the progress of various infrastructure construction projects in Beijing. These scenarios affected the pressure on the soft land base in Beijing and reduced the surface subsidence trend to some extent. © 2023 by the authors.

Discovery of DNA aptamers targeting SARS-CoV-2 nucleocapsid protein and protein-binding epitopes for label-free COVID-19 diagnostics.

The spread of COVID-19 has affected billions of people across the globe, and the diagnosis of viral infection still needs improvement. Because of high immunogenicity and abundant expression during viral infection, SARS-CoV-2 nucleocapsid (N) protein could be an important diagnostic marker. This study aimed to develop a label-free optical aptasensor fabricated with a novel single-stranded DNA aptamer to detect the N protein. The N-binding aptamers selected using asymmetric-emulsion PCR-SELEX and their binding affinity and cross-reactivity were characterized by biolayer interferometry. The tNSP3 aptamer (44 nt) was identified to bind the N protein of wild type and Delta and Omicron variants with high affinity (KD in the range of 0.6-3.5 nM). Utilizing tNSP3 to detect the N protein spiked in human saliva evinced the potential of this aptamer with a limit of detection of 4.5 nM. Mass spectrometry analysis was performed along with molecular dynamics simulation to obtain an insight into how tNSP3 binds to the N protein. The identified epitope peptides are localized within the RNA-binding domain and C terminus of the N protein. Hence, we confirmed the performance of this aptamer as an analytical tool for COVID-19 diagnosis.

Diffractive Phase Interferometry (DPI), CRP and COVID-19 RT-PCR Tests in COVID-19 Patients with Different Ages in AJMAN, UAE: A Comparative Study

Background: Fast and accurate diagnosis plays an important role in controlling and further preventing COVID-19. This study was conducted in the Thumbay laboratory of Gulf Medical University (Ajman, UAE) to assess the correlations between DPI (Diffractive Phase Interferometry), COVID-19 RT-PCR, and CRP tests in COVID-19 patients of different ages and to compare the effectiveness of each parameter. Methods and Results: A cross-sectional analytic study was conducted among 150 patients diagnosed with COVID-19 who were admitted to the Thumbay University Hospital. Their general data was collected from the LDM system, and from among the suspected patients who came to do the RT-PCR test, 230 were selected as volunteers to participate in this study, and further laboratory tests like CRP level and DPI test were done for them. The nasal swab was collected for a PCR test. Out of 230 nasal swab samples, 150 were positive and 80 were negative for SARS-CoV-2 RNA by real-time RT-PCR assay. Among the 150 positive RT-PCR, 90 false negative DPI tests were from a sample with a high real-time RT-PCR. While 60 true positive DPI tests were positive real-time RT-PCR for swab specimens. Among the 80 negative RT-PCR, 79 were true negative and 1 was a false positive. The predictive positive value of the DPI test was 40% and the predictive negative value of the test was 98%. DPI has at least one tie between the positive actual state group and the negative actual state group. The results show weak and moderate positive correlations between CRP and the age groups. Conclusion: The combined detection of the three indicators (RT-PCR, DPI, and CRP) are positively related to COVID-19 infection;therefore, these indicators will enable effective intervention measures to be implemented in time and the rates of severe illness and mortality to be reduced. © 2022, International Medical Research and Development Corporation. All rights reserved.

Functional studies on the interaction between anti-PF4 antibodies and anticoagulants in vaccine-induced thrombotic thrombocytopenia

Background: With increasing number of vaccinations against SARS-CoV-2, rare but life threatening thrombotic events at unusual sites have been reported, and collectively this phenomenon is termed as vaccine-induced immune thrombotic thrombocytopenia (VITT). Pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT), and associated with platelet-activating antibodies against platelet factor 4 (PF4). Aim(s): Current guidelines for anticoagulation in VITT patients are issued accordingly, with a focus on non-heparin anticoagulants. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux and argatroban with VITT-Ab/ PF4 complexes. Method(s): We utilized an in-house enzyme immunoassays (EIA) to estimate antibody binding, inhibition and dissociation of preformed PF4-VITT complexes. Using biolayer interferometry (BLI), we analyzed binding kinetics and dissociation of complexes in real time. In a flow-based ex vivo model, we assessed the impact of anticoagulants on VITT-mediated thrombus formation. Result(s): We found that heparin and danaparoid not only inhibited VITT IgG binding to PF4 but were also able to effectively dissociate preformed PF4/IgG complexes in EIA. In BLI, binding of PF4 specific antibodies was observed for all VITT samples tested, and we found remarkable changes in their dissociation after addition of various anticoagulants. Furthermore, IgGs from VITT patients induce increased thrombus formation in comparison to the healthy controls (mean % SAC +/- SEM: 11.59 +/- 0.57 vs. 1.99 +/- 0.34 respectively, p < 0.001), which can further be effectively inhibited with danaparoid and heparin (mean % SAC +/- SEM 2.82 +/- 0.50 and 1.85 +/- 0.56. p < 0.001). Fondaparinux and argatroban inhibited thrombus formation;however, they did not affect antibody binding. Conclusion(s): Taken together, our data shed a light on suitability of anticoagulants in VITT, and indicate that negatively charged anticoagulants can disrupt VITT-Ab/ PF4 interactions, which might serve as an approach to reduce antibody-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulation in VITT patients could be made.

A Sulfated Abalone Polysaccharide Inhibited SARS-CoV-2 Infection of Vero E6 Cells In Vitro.

Sulfate polysaccharides, such as heparin sulfate, have been found to have inhibitory activity against SARS-CoV-2. An abalone polysaccharide, AGSP, was deeply sulfate modified using the chlorosulfonic acid/pyridine method, yielding S-AGSP. AGSP and S-AGSP inhibitions of SARS-CoV-2 infection of Vero E6 cells were tested in vitro. The interference of AGSP or S-AGSP on the binding interaction between the SARS-CoV-2 spike protein and angiotensin-converting enzyme was tested using a biolayer interferometry assay. Results showed that S-AGSP, above a concentration of 1.87 µg/mL, significantly inhibited SARS-CoV-2 infection of Vero E6 cells. Compared with AGSP, S-AGSP obviously weakened the affinity between the SARS-CoV-2 spike protein and ACE2. The polysaccharide's sulfate content played a vital role in influencing the binding affinity of spike protein to ACE2. Therefore, S-AGSP has potential as a COVID-19 competitive inhibitor as well as a candidate to be repurposed as a prophylactic COVID-19 therapeutic.

Optical Label-Free Method for Characterization of Kinetics of Antibodies to SARS-CoV-2

To date, a dangerous infectious disease of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-Co V-2) has been a significant source of morbidity and high mortality worldwide. In this work, an optical label-free method is developed for measurements and characterization of kinetics of antibodies to SARS-CoV-2 in real time. A biomarker of COVID-19, namely, spike protein of SARS-CoV-2 antigen was used as a model antigen. The proposed method employs inexpensive and widely available consumables compatible with various chemical interfaces. It is promising for assessment the kinetics of humoral response to SARS-CoV-2 infection or postvaccination. © 2022 IEEE.

Directly immersible silicon photonic probes: Application to rapid SARS-CoV-2 serological testing.

Silicon photonic probes based on broad-band Mach-Zehnder interferometry are explored for the first time as directly immersible immunosensors alleviating the need for microfluidics and pumps. Each probe includes two U-shaped waveguides allowing light in- and out-coupling from the same chip side through a bifurcated fiber and a mechanical coupler. At the opposite chip side, two Mach-Zehnder interferometers (MZI) are located enabling real-time monitoring of binding reactions by immersion of this chip side into a sample. The sensing arm windows of the two MZIs have different length resulting in two distinct peaks in the Fourier domain, the phase shift of which can be monitored independently through Fast Fourier Transform of the output spectrum. The photonic probes analytical potential was demonstrated through detection of antibodies against SARS-CoV-2 in human serum samples. For this, one MZI was functionalized with the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike 1 protein, and the other with bovine serum albumin to serve as reference. The biofunctionalized probes were immersed for 10 min in human serum sample and then for 5 min in goat anti-human IgG Fc specific antibody solution. Using a humanized rat antibody against SARS-CoV-2 RBD, a detection limit of 20 ng/mL was determined. Analysis of human serum samples indicated that the proposed sensor discriminated completely non-infected/non-vaccinated from vaccinated individuals, and the antibodies levels determined correlated well with those determined in the same samples by ELISA. These results demonstrated the potential of the proposed sensor to serve as an efficient tool for expeditious point-of-care testing.

Photonic resonator interferometric scattering microscopy

Interferometric scattering microscopy is a newly emerging alternative to fluorescence microscopy in biomedical research and diagnostic testing due to its ability to detect nano-objects such as individual proteins, extracellular vesicles, and virions individually through their intrinsic elastic light scattering. To improve the signal-to-noise ratio, we developed photonic resonator interferometric scattering microscopy (PRISM) in which a photonic crystal (PC) resonator is used as the sample substrate. The scattered light is amplified by the PC through resonant near-field enhancement, which then interferes with the <1% transmitted light to create intensity contrast. Importantly, the scattered photons assume the wavevectors defined by PC's photonic band structure, resulting in the ability to utilize a non-immersion objective without significant loss at illumination density as low as 25 W/cm2. We demonstrate virus and protein detection, including highly selective capture and counting of intact pseudotype SARS-CoV-2 from saliva with sensitivity equivalent to conventional nucleic acid tests. The results showcase the promise of nanophotonic surfaces in the development of resonance-enhanced interferometric microscopies, and as a single step, room temperature, and rapid viral detection technology. © 2022 SPIE.

ANTIGENICITY of the MU (B.1.621) and A.2.5 SARS-CoV-2 SPIKES

Background: The rapid emergence of SARS-CoV-2 variants is fueling the recent waves of the COVID-19 pandemic. Recently identified Mu (B.1.621) and A.2.5 variants carry some mutations shared by other variants of concerns (VOCs). For example, N501Y and E484K mutations in the receptor-binding domain (RBD) domain detected in B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) are now present within the Mu variant. Similarly, the L452R mutation of B.1.617.2 (Delta) variant is now present in A.2.5. Here, we evaluated the capacity of Mu and A.2.5 Spikes to interact with angiotensin-converting enzyme 2 (ACE2) and performed binding and neutralization assays with plasma from vaccinated individuals. In addition, to better understand their antigenic properties, we compared both Mu and A.2.5 with Alpha, Beta, Gamma and Delta VOCs Spikes. Methods: Cells expressing the different Spikes were interrogated for their capacity to interact with the ACE2 receptor using a recombinant ACE2-Fc recombinant protein. We also evaluated their recognition by plasma from BNT162b2 vaccinated individuals. Biolayer interferometry (BLI) was used to measure the binding kinetics of selected RBD mutants to soluble ACE2 (sACE2). Finally, we evaluated the susceptibility of pseudoviral particles bearing the different Spikes to neutralization by plasma from vaccinated individuals. Results: All SARS-CoV-2 S-glycoprotein variants were recognized less efficiently by plasma from vaccinated SARS-CoV-2 naïve and previously-infected individuals compared to D614G Spike with the exception of B.1.1.7 S-glycoprotein. Enhanced ACE2 interaction by the Spikes tested was associated with a decrease in the off-rate of the ACE2-RBD interaction. Pseudoviral particles bearing the Spike of Mu variant were similarly neutralized by plasma from vaccinated individuals than those carrying the Beta and Delta Spikes. Conclusion: Plasma from vaccinated SARS-CoV-2 naïve and previously-infected individuals efficiently recognized all the Spikes tested. The decreased neutralization susceptibility of pseudoviral particles expressing the Mu Spike was similar to Beta and Delta, thus underscoring the importance of functionally tracking emerging variants. In summary, our results highlight the importance of measuring critical parameters such as ACE2 interaction, plasma recognition and neutralization from each emerging variant.

Lactoferrin Inhibition of the Complex Formation between ACE2 Receptor and SARS CoV-2 Recognition Binding Domain.

The present investigation focuses on the analysis of the interactions among human lactoferrin (LF), SARS-CoV-2 receptor-binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2) receptor in order to assess possible mutual interactions that could provide a molecular basis of the reported preventative effect of lactoferrin against CoV-2 infection. In particular, kinetic and thermodynamic parameters for the pairwise interactions among the three proteins were measured via two independent techniques, biolayer interferometry and latex nanoparticle-enhanced turbidimetry. The results obtained clearly indicate that LF is able to bind the ACE2 receptor ectodomain with significantly high affinity, whereas no binding to the RBD was observed up to the maximum "physiological" lactoferrin concentration range. Lactoferrin, above 1 µM concentration, thus appears to directly interfere with RBD-ACE2 binding, bringing about a measurable, up to 300-fold increase of the KD value relative to RBD-ACE2 complex formation.

Rapid Optical Biosensing of SARS-CoV-2 Spike Proteins in Artificial Samples.

Tests for SARS-CoV-2 are crucial for the mass surveillance of the incidence of infection. The long waiting time for classic nucleic acid test results highlights the importance of developing alternative rapid biosensing methods. Herein, we propose a fiber-optic biolayer interferometry-based biosensor (FO-BLI) to detect SARS-CoV-2 spike proteins, extracellular domain (ECD), and receptor-binding domain (RBD) in artificial samples in 13 min. The FO-BLI biosensor utilized an antibody pair to capture and detect the spike proteins. The secondary antibody conjugated with horseradish peroxidase (HRP) reacted with the enzyme substrate for signal amplification. Two types of substrates, 3,3'-diaminobenzidine (DAB) and an advanced 3-Amino-9-ethylcarbazole (i.e., AMEC), were applied to evaluate their capabilities in enhancing signals and reaching high sensitivity. After careful comparison, the AMEC-based FO-BLI biosensor showed better assay performance, which detected ECD at a concentration of 32-720 pM and RBD of 12.5-400 pM in artificial saliva and serum, respectively. The limit of detection (LoD) for SARS-CoV-2 ECD and RBD was defined to be 36 pM and 12.5 pM, respectively. Morphology of the metal precipitates generated by the AMEC-HRP reaction in the fiber tips was observed using field emission scanning electron microscopy (SEM). Collectively, the developed FO-BLI biosensor has the potential to rapidly detect SARS-CoV-2 antigens and provide guidance for "sample-collect and result-out on-site" mode.

Applications of Surface Plasmon Resonance and Biolayer Interferometry for Virus-Ligand Binding.

Surface plasmon resonance and biolayer interferometry are two common real-time and label-free assays that quantify binding events by providing kinetic parameters. There is increased interest in using these techniques to characterize whole virus-ligand interactions, as the methods allow for more accurate characterization than that of a viral subunit-ligand interaction. This review aims to summarize and evaluate the uses of these technologies specifically in virus-ligand and virus-like particle-ligand binding cases to guide the field towards studies that apply these robust methods for whole virus-based studies.

Measurements of SARS-CoV-2 antibody dissociation rate constant by chaotrope-free biolayer interferometry in serum of COVID-19 convalescent patients.

Kinetics measurements of antigen-antibody binding interactions are critical to understanding the functional efficiency of SARS-CoV-2 antibodies. Previously reported chaotrope-based avidity assays that rely on artificial disruption of binding do not reflect the natural binding kinetics. This study developed a chaotrope- and label-free biolayer interferometry (BLI) assay for the real-time monitoring of receptor binding domain (RBD) binding kinetics with SARS-CoV-2 spike protein in convalescent COVID-19 patients. An improved conjugation biosensor probe coated with streptavidin-polysaccharide (SA-PS) led to a six-fold increase of signal intensities and two-fold reduction of non-specific binding (NSB) compared to streptavidin only probe. Furthermore, by utilizing a separate reference probe and biotin-human serum albumin (B-HSA) blocking process to subtracted NSB signal in serum, this BLI biosensor can measure a wide range of the dissociation rate constant (koff), which can be measured without knowledge of the specific antibody concentrations. The clinical utility of this improved BLI kinetics assay was demonstrated by analyzing the koff values in sera of 24 pediatric (≤18 years old) and 63 adult (>18 years old) COVID-19 convalescent patients. Lower koff values for SARS-CoV-2 serum antibodies binding to RBD were measured in samples from children. This rapid, easy to operate and chaotrope-free BLI assay is suitable for clinical use and can be readily adapted to characterize SARS-CoV-2 antibodies developed by COVID-19 patients and vaccines.

Exploring SARS-CoV-2 Delta variant spike protein receptor-binding domain (RBD) as a target for tanshinones and antimalarials.

The interaction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) of spike protein with angiotensin-converting enzyme 2 (ACE2) mediates cell invasion. While this interaction mechanism is conserved, the RBD is affected by amino acid mutations in variants such as Delta and Omicron, resulting in enhanced transmissibility and altered ligand binding. Tanshinones are currently investigated as multi-target antiviral agents, but the studies were limited to the original SARS-CoV-2. This study aims at investigating the interaction of tanshinones with the Delta RBD. Chloroquine, methylene blue and pyronaridine, antimalarials previously identified as SARS-CoV-2 RBD binders, were studied for reference. Docking indicated the best scores for tanshinones, while bio-layer interferometry and molecular dynamics highlighted methylene blue as the best Delta RBD binder, although with decreased affinity with respect to the original strain.

Holistic Approach to R&D Products’ Evaluation for Commercialization under Open Innovations

A holistic approach to R&D products’ evaluation for commercialization under open innovations is developed. The approach is tested on the example of the device of the interferometric determination of the refractive index of crystalline materials in the optical range. The proposed approach will allow setting a price that will satisfy all the parties of a transfer agreement with a higher level of accuracy and will meet market requirements. Unlike popular methods of evaluating the R&D product, a holistic approach will, on the one hand, be based on the actual costs and the break-even level of a R&D product and, on the other hand, will determine how much the consumer is receptive to a R&D product, and, then again, will show how the added value of the product will develop under the influence of market effects. It is noted that the application of a holistic approach to R&D products’ evaluation for commercialization should be supplemented by assessing the willingness of potential customers to purchase this R&D product at a specific price. It is proved that the proposed holistic approach to R&D products’ evaluation for commercialization is multifunctional. The approach can be applied to different types of economic activity, R&D products, and types of markets. The obtained prices based on the application of a holistic approach to R&D products’ evaluation for commercialization and the results of marketing research of the interferometry market testified to potential prospects of the commercialization of a R&D product of the interferometric determination of the refractive index of crystalline materials in the optical range and its long-term competitiveness. Based on the results, the key provisions of the concept of providing competitive benefits for the period of implementation of the analyzed R&D product are identified. Taking into account fundamental elements of the open innovation paradigm underpins the authors’ holistic approach.