Immunoaffinity biosensors for the detection of SARS-CoV-1 using screened Fv-antibodies from an autodisplayed Fv-antibody library.

The detection of severe acute respiratory syndrome coronavirus (SARS-CoV-1) was demonstrated using screened Fv-antibodies for SPR biosensor and impedance spectrometry. The Fv-antibody library was first prepared on the outer membrane of E. coli using autodisplay technology and the Fv-variants (clones) with a specific affinity toward the SARS-CoV-1 spike protein (SP) were screened using magnetic beads immobilized with the SP. Upon screening the Fv-antibody library, two target Fv-variants (clones) with a specific binding affinity toward the SARS-CoV-1 SP were determined and the Fv-antibodies on two clones were named "Anti-SP1" (with CDR3 amino acid sequence: 1GRTTG5NDRPD11Y) and "Anti-SP2" (with CDR3 amino acid sequence: 1CLRQA5GTADD11V). The binding affinities of the two screened Fv-variants (clones) were analyzed using flow cytometry and the binding constants (KD) were estimated to be 80.5 ± 3.6 nM for Anti-SP1 and 45.6 ± 8.9 nM for Anti-SP2 (n = 3). In addition, the Fv-antibody including three CDR regions (CDR1, CDR2, and CDR3) and frame regions (FRs) between the CDR regions was expressed as a fusion protein (Mw. 40.6 kDa) with a green fluorescent protein (GFP) and the KD values of the expressed Fv-antibodies toward the SP estimated to be 15.3 ± 1.5 nM for Anti-SP1 (n = 3) and 16.3 ± 1.7 nM for Anti-SP2 (n = 3). Finally, the expressed Fv-antibodies screened against SARS-CoV-1 SP (Anti-SP1 and Anti-SP2) were applied for the detection of SARS-CoV-1. Consequently, the detection of SARS-CoV-1 was demonstrated to be feasible using the SPR biosensor and impedance spectrometry utilizing the immobilized Fv-antibodies against the SARS-CoV-1 SP.

IDWeek 2022

IDWeek is the joint annual meeting of the Infectious Diseases Society of America (IDSA), Society for Healthcare Epidemiology of America (SHEA), the HIV Medicine Association (HIVMA), the Pediatric Infectious Diseases Society (PIDS) and the Society of Infectious Diseases Pharmacists (SIDP). For the first time since the COVID-19 public health emergency began, IDWeek 2022 returned to in-person attendance. It was held in Washington, D.C., and the meeting comprised 5 days of live sessions and on-demand content that included posters and oral presentations.Copyright © 2023 Clarivate.

Virtual Screening-Based Peptides Targeting Spike Protein to Inhibit Porcine Epidemic Diarrhea Virus (PEDV) Infection.

Due to the rapid mutation of porcine epidemic diarrhea virus (PEDV), existing vaccines cannot provide sufficient immune protection for pigs. Therefore, it is urgent to design the affinity peptides for the prevention and control of this disease. In this study, we made use of a molecular docking technology for virtual screening of affinity peptides that specifically recognized the PEDV S1 C-terminal domain (CTD) protein for the first time. Experimentally, the affinity, cross-reactivity and sensitivity of the peptides were identified by an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR) test, separately. Subsequently, Cell Counting Kit-8 (CCK-8), quantitative real-time PCR (qRT-PCR), Western blot and indirect immunofluorescence were used to further study the antiviral effect of different concentrations of peptide 110766 in PEDV. Our results showed that the P/N value of peptide 110766 at 450 nm reached 167, with a KD value of 216 nM. The cytotoxic test indicated that peptide 110766 was not toxic to vero cells. Results of the absolute quantitative PCR revealed that different concentrations (3.125 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, 100 µM, 200 µM) of peptide 110766 could significantly reduce the viral load of PEDV compared with the virus group (p < 0.0001). Similarly, results of Western blot and indirect immunofluorescence also suggested that the antiviral effect of peptide 110766 at 3.125 is still significant. Based on the above research, high-affinity peptide 110766 binding to the PEDV S1-CTD protein was attained by a molecular docking technology. Therefore, designing, screening, and identifying affinity peptides can provide a new method for the development of antiviral drugs for PEDV.

Gradient method for accurate affinity determinations.

The value of the affinity constants (kd, ka, and KD) that are determined by label free interaction analysis methods are strongly affected by the ligand density at the sensor surface [1]. This paper outlines a new SPR-imaging method that applies a ligand density gradient enabling the analyte response to be extrapolated to Rmax = 0 µRIU. The mass transport limited region is used to determine the analyte concentration. Cumbersome optimization procedures for tuning the ligand density is prevented and surface dependent effects as rebinding, strong biphasic behavior etcetera are minimized. The method can be fully automated for e.g. accurate determination of the quality of antibodies from commercial sources.

Ultrasensitive voltammetric detection of SARS-CoV-2 in clinical samples.

In every pandemic, it is critical to test as many people as possible and keep track of the number of new cases of infection. Therefore, there is a need for novel, fast and unambiguous testing methods. In this study, we designed a sandwich-type voltammetric immunosensor based on unlabeled- and labeled with a redox probe antibodies against virus spike protein for fast and ultrasensitive detection of SARS-CoV-2. The process of the preparation of the sensor layer included chemisorption of cysteamine layer and covalent anchoring of antibody specific for the S1 subunit of the S protein. The source of the voltametric signal was the antibody labeled with the redox probe, which was introduced onto biosensor surface only after the recognition of the virus. This easy-to-handle immunosensor was characterized by a wide analytical range (2.0·10-7 to 0.20 mg·L-1) and low detection limit (8.0·10-8 mg·L-1 ≡ 0.08 pg·mL-1 ≡ 4 virions·µL-1). The utility of the designed device was also evidenced by the detection of SARS-CoV-2 in the clinical samples. Moreover, the main advantage and a huge novelty of the developed device, compared to those already existing, is the moment of generating the analytical signal of the redox probe that appears only after the virus recognition. Thus, our diagnostic innovation may considerably contribute to controlling the COVID-19 pandemic. The as-developed immunosensor may well offer a novel alternative approach for viral detection that could complement or even replace the existing methods.

IDWeek 2022 Washington, DC - October 19-23, 2022

IDWeek is the joint annual meeting of the Infectious Diseases Society of America (IDSA), Society for Healthcare Epidemiology of America (SHEA), the HIV Medicine Association (HIVMA), the Pediatric Infectious Diseases Society (PIDS) and the Society of Infectious Diseases Pharmacists (SIDP). For the first time since the COVID-19 public health emergency began, IDWeek 2022 returned to in-person attendance. It was held in Washington, D.C., and the meeting comprised 5 days of live sessions and on-demand content that included posters and oral presentations.

Impedimetric Nanobiosensor for the Detection of SARS-CoV-2 Antigens and Antibodies.

Detection of antigens and antibodies (Abs) is of great importance in determining the infection and immunity status of the population, as they are key parameters guiding the handling of pandemics. Current point-of-care (POC) devices are a convenient option for rapid screening; however, their sensitivity requires further improvement. We present an interdigitated gold nanowire-based impedance nanobiosensor to detect COVID-19-associated antigens (receptor-binding domain of S1 protein of the SARS-CoV-2 virus) and respective Abs appearing during and after infection. The electrochemical impedance spectroscopy technique was used to assess the changes in measured impedance resulting from the binding of respective analytes to the surface of the chip. After 20 min of incubation, the sensor devices demonstrate a high sensitivity of about 57 pS·sn per concentration decade and a limit of detection (LOD) of 0.99 pg/mL for anti-SARS-CoV-2 Abs and a sensitivity of around 21 pS·sn per concentration decade and an LOD of 0.14 pg/mL for the virus antigen detection. Finally, the analysis of clinical plasma samples demonstrates the applicability of the developed platform to assist clinicians and authorities in determining the infection or immunity status of the patients.

Inhibitory activity of a sulfated oligo-porphyran from Pyropia yezoensis against SARS-CoV-2.

COVID-19 caused by SARS-CoV-2 has spread around the world at an unprecedented rate. A more homogeneous oligo-porphyran with mean molecular weight of 2.1 kD, named OP145, was separated from Pyropia yezoensis. NMR analysis showed OP145 was mainly composed of →3)-ß-d-Gal-(1 â†’ 4)-α-l-Gal (6S) repeating units with few replacement of 3,6-anhydride, and the molar ratio was 1:0.85:0.11. MALDI-TOF MS revealed OP145 contained mainly tetrasulfate-oligogalactan with Dp range from 4 to 10 and with no more than two 3,6-anhydro-α-l-Gal replacement. The inhibitory activity of OP145 against SARS-CoV-2 was investigated in vitro and in silico. OP145 could bind to Spike glycoprotein (S-protein) through SPR result, and pseudovirus tests confirmed that OP145 could inhibite the infection with an EC50 of 37.52 µg/mL. Molecular docking simulated the interaction between the main component of OP145 and S-protein. All the results indicated that OP145 had the potency to treat and prevent COVID-19.

Protocol to determine antibody affinity and concentration in complex solutions using microfluidic antibody affinity profiling.

Conventional methods of measuring affinity are limited by artificial immobilization, large sample volumes, and homogeneous solutions. This protocol describes microfluidic antibody affinity profiling on complex human samples in solution to obtain a fingerprint reflecting both affinity and active concentration of the target protein. To illustrate the protocol, we analyze the antibody response in SARS-CoV-2 omicron-naïve samples against different SARS-CoV-2 variants of concern. However, the protocol and the technology are amenable to a broad spectrum of biomedical questions. For complete details on the use and execution of this protocol, please refer to Emmenegger et al. (2022),1 Schneider et al. (2022),2 and Fiedler et al. (2022).3.

Novel Affibody Molecules Specifically Bind to SARS-CoV-2 Spike Protein and Efficiently Neutralize Delta and Omicron Variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented public health disaster in human history, and its spike (S) protein is the major target for vaccines and antiviral drug development. Although widespread vaccination has been well established, the viral gene is prone to rapid mutation, resulting in multiple global spread waves. Therefore, specific antivirals are needed urgently, especially those against variants. In this study, the domain of the receptor binding motif (RBM) and fusion peptide (FP) (amino acids [aa] 436 to 829; denoted RBMFP) of the SARS-CoV-2 S protein was expressed as a recombinant RBMFP protein in Escherichia coli and identified as being immunogenic and antigenically active. Then, the RBMFP proteins were used for phage display to screen the novel affibody. After prokaryotic expression and selection, four novel affibody molecules (Z14, Z149, Z171, and Z327) were obtained. Through surface plasmon resonance (SPR) and pseudovirus neutralization assay, we showed that affibody molecules specifically bind to the RBMFP protein with high affinity and neutralize against SARS-CoV-2 pseudovirus infection. Especially, Z14 and Z171 displayed strong neutralizing activities against Delta and Omicron variants. Molecular docking predicted that affibody molecule interaction sites with RBM overlapped with ACE2. Thus, the novel affibody molecules could be further developed as specific neutralization agents against SARS-CoV-2 variants. IMPORTANCE SARS-CoV-2 and its variants are threatening the whole world. Although a full dose of vaccine injection showed great preventive effects and monoclonal antibody reagents have also been used for a specific treatment, the global pandemic persists. So, developing new vaccines and specific agents are needed urgently. In this work, we expressed the recombinant RBMFP protein as an antigen, identified its antigenicity, and used it as an antigen for affibody phage-display selection. After the prokaryotic expression, the specific affibody molecules were obtained and tested for pseudovirus neutralization. Results showed that the serum antibody induced by RBMFP neutralized Omicron variants. The screened affibody molecules specifically bound the RBMFP of SARS-CoV-2 with high affinity and neutralized the Delta and Omicron pseudovirus in vitro. So, the RBMFP induced serum provides neutralizing effects against pseudovirus in vitro, and the affibodies have the potential to be developed into specific prophylactic agents for SARS-CoV-2 and its variants.

Exploring novel targets of sitagliptin for type 2 diabetes mellitus: Network pharmacology, molecular docking, molecular dynamics simulation, and SPR approaches.

Background: Diabetes has become a serious global public health problem. With the increasing prevalence of type 2 diabetes mellitus (T2DM), the incidence of complications of T2DM is also on the rise. Sitagliptin, as a targeted drug of DPP4, has good therapeutic effect for T2DM. It is well known that sitagliptin can specifically inhibit the activity of DPP4 to promote insulin secretion, inhibit islet ß cell apoptosis and reduce blood glucose levels, while other pharmacological mechanisms are still unclear, such as improving insulin resistance, anti-inflammatory, anti-oxidative stress, and anti-fibrosis. The aim of this study was to explore novel targets and potential signaling pathways of sitagliptin for T2DM. Methods: Firstly, network pharmacology was applied to find the novel target most closely related to DPP4. Semi-flexible molecular docking was performed to confirm the binding ability between sitagliptin and the novel target, and molecular dynamics simulation (MD) was carried to verify the stability of the complex formed by sitagliptin and the novel target. Furthermore, surface-plasmon resonance (SPR) was used to explored the affinity and kinetic characteristics of sitagliptin with the novel target. Finally, the molecular mechanism of sitagliptin for T2DM was predicted by the enrichment analysis of GO function and KEGG pathway. Results: In this study, we found the cell surface receptor-angiotensin-converting enzyme 2 (ACE2) most closely related to DPP4. Then, we confirmed that sitagliptin had strong binding ability with ACE2 from a static perspective, and the stability of sitagliptin-ACE2 complex had better stability and longer binding time than BAR708-ACE2 in simulated aqueous solution within 50 ns. Significantly, we have demonstrated a strong affinity between sitagliptin and ACE2 on SPR biosensor, and their kinetic characteristics were "fast binding/fast dissociation". The guiding significance of clinical administration: low dose can reach saturation, but repeated administration was needed. Finally, there was certain relationship between COVID-19 and T2DM, and ACE2/Ang-(1-7)/Mas receptor (MasR) axis may be the important pathway of sitagliptin targeting ACE2 for T2DM. Conclusion: This study used different methods to prove that ACE2 may be another novel target of sitagliptin for T2DM, which extended the application of ACE2 in improving diabetes mellitus.

Numerical Analysis of Coronavirus Detection Using Photonic Crystal Fibre-Based SPR Sensor.

Coronavirus disease (COVID-19) is a worldwide health emergency caused by the coronavirus 2 (severe acute respiratory illness) (SARS-CoV-2). COVID-19 has a wide range of symptoms, making a definitive diagnosis difficult. The shortage of equipment for testing technology COVID-19 has resulted in long queues for COVID-19 testing, which is a major problem. COVID-19 testing is currently performed using sluggish and costly technology like single-photon emission computed tomography (SPECT), computed tomography (CT), positron emission tomography (PET), and enzyme-linked immunosorbent assay (ELISA). The gold standard test for diagnosing COVID-19 is real-time reverse transcriptase-polymerase chain reaction (RT-PCR), which necessitates highly skilled workers and has a lengthy turnaround time. However, rapid and affordable immunodiagnostic techniques (antigen or antibody tests) are also available with some trade off accuracy. Optical sensors are frequently employed in a variety of applications, because of their increased sensitivity, strong selectivity, rapid reaction times, and outstanding resolution. The use of photonic crystal fibre (PCF) is advantageous for the quick detection of the new coronavirus and is suggested with the use of a PCF-based (Au/BaTiO3/graphene) multilayered surface plasmon resonance (SPR) biosensor. The proposed sensor can quickly detect the COVID-19 virus in two different ligand-analyte environments: (i) the virus spike receptor-binding domain (RBD) as an analyte and monoclonal antibodies (mAbs) as a probe ligand, and (ii) monoclonal antibodies (IgG or IgM) as an analyte and the virus spike RBD as a probe ligand. The finite element method (FEM) is used to quantitatively examine the performance of the PCF-based multilayered SPR sensor.

Development of a Single-Chain Variable Fragment of CR3022 for a Plasmonic-Based Biosensor Targeting the SARS-CoV-2 Spike Protein.

Two years after SARS-CoV-2 caused the first case of COVID-19, we are now in the "new normal" period, where people's activity has bounced back, followed by the easing of travel policy restrictions. The lesson learned is that the wide availability of accurate and rapid testing procedures is crucial to overcome possible outbreaks in the future. Therefore, many laboratories worldwide have been racing to develop a new point-of-care diagnostic test. To aid continuous innovation, we developed a plasmonic-based biosensor designed explicitly for portable Surface Plasmon Resonance (SPR). In this study, we designed a single chain variable fragment (scFv) from the CR3022 antibody with a particular linker that inserted a cysteine residue at the second position. It caused the linker to have a strong affinity to the gold surface through thiol-coupling and possibly become a ready-to-use bioreceptor toward a portable SPR gold chip without purification steps. The theoretical affinity of this scFv on spike protein was -64.7 kcal/mol, computed using the Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method from the 100 ns molecular dynamics trajectory. Furthermore, the scFv was produced in Escherichia coli BL21 (DE3) as a soluble protein. The binding activity toward Spike Receptor Binding Domain (RBD) SARS-CoV-2 was confirmed with a spot-test, and the experimental binding free energy of -10.82 kcal/mol was determined using portable SPR spectroscopy. We hope this study will be useful in designing specific and low-cost bioreceptors, particularly early in an outbreak when the information on antibody capture is still limited.

Development and Application of an SPR Nanobiosensor Based on AuNPs for the Detection of SARS-CoV-2 on Food Surfaces.

A new transmission route of SARS-CoV-2 through food was recently considered by the World Health Organization (WHO), and, given the pandemic scenario, the search for fast, sensitive, and low-cost methods is necessary. Biosensors have become a viable alternative for large-scale testing because they overcome the limitations of standard techniques. Herein, we investigated the ability of gold spherical nanoparticles (AuNPs) functionalized with oligonucleotides to detect SARS-CoV-2 and demonstrated their potential to be used as plasmonic nanobiosensors. The loop-mediated isothermal amplification (LAMP) technique was used to amplify the viral genetic material from the raw virus-containing solution without any preparation. The detection of virus presence or absence was performed by ultraviolet-visible (UV-Vis) absorption spectroscopy, by monitoring the absorption band of the surface plasmonic resonance (SPR) of the AuNPs. The displacement of the peak by 525 nm from the functionalized AuNPs indicated the absence of the virus (particular region of gold). On the other hand, the region ~300 nm indicated the presence of the virus when RNA bound to the functionalized AuNPs. The nanobiosensor system was designed to detect a region of the N gene in a dynamic concentration range from 0.1 to 50 × 103 ng·mL-1 with a limit of detection (LOD) of 1 ng·mL-1 (2.7 × 103 copy per µL), indicating excellent sensitivity. The nanobiosensor was applied to detect the SARS-CoV-2 virus on the surfaces of vegetables and showed 100% accuracy compared to the standard quantitative reverse transcription polymerase chain reaction (RT-qPCR) technique. Therefore, the nanobiosensor is sensitive, selective, and simple, providing a viable alternative for the rapid detection of SARS-CoV-2 in ready-to-eat vegetables.

A Four-Channel Surface Plasmon Resonance Sensor Functionalized Online for Simultaneous Detections of Anti-SARS-CoV-2 Antibody, Free Viral Particles, and Neutralized Viral Particles.

Current tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detect either the constituent nucleic acids/proteins of the viral particles or antibodies specific to the virus, but cannot provide information about viral neutralization by an antibody and the efficacy of an antibody. Such information is important about individuals' vulnerability to severe symptoms or their likelihood of showing no symptoms. We immobilized online SARS-CoV-2 spike (S1) protein and angiotensin-converting enzyme 2 (ACE2) into separate surface plasmon resonance (SPR) channels of a tris-nitrilotriacetic acid (tris-NTA) chip to simultaneously detect the anti-S1 antibody and viral particles in serum samples. In addition, with a high-molecular-weight-cutoff filter, we separated the neutralized viral particles from the free antibody molecules and used a sensing channel immobilized with Protein G to determine antibody-neutralized viral particles. The optimal density of probe molecules in each fluidic channel can be precisely controlled through the closure and opening of the specific ports. By utilizing the high surface density of ACE2, multiple assays can be carried out without regenerations. These three species can be determined with a short analysis time (<12 min per assay) and excellent sensor-to-sensor/cycle-to-cycle reproducibility (RSD < 5%). When coupled with an autosampler, continuous assays can be performed in an unattended manner at a single chip for up to 6 days. Such a sensor capable of assaying serum samples containing the three species at different levels provides additional insights into the disease status and immunity of persons being tested, which should be helpful for containing the SARS-CoV-2 spread during the era of incessant viral mutations.

Successful creation of a home spirometry program within an adult cystic fibrosis clinic

Background: Highly effective modulator therapy and the COVID-19 pandemic have transformed cystic fibrosis (CF) care. Increased use of telehealth has resulted in fewer in-person visits, although close monitoring continues to be crucial in preventing complications. Home spirometry is a useful, accessible tool that people with CF can use to monitor their lung function. In spring 2020, the University of North Carolina (UNC) Adult CF Clinic began to distribute home spirometers to their patients through the Cystic Fibrosis Foundation (CFF). At the time, we lacked a standardized home spirometry program and found it challenging to engage patients in regular use of the device. As a result, the UNC adult CF advanced practice provider (APP) and respiratory therapist (RT) collaborated to lead a quality improvement (QI) project aimed at improving home spirometry at their center. Method(s): Goals of the QI effort were to provide individual education for all patients with a home spirometer, incorporate home spirometry assessments into the RT workflow, and provide monthly interpretation of home spirometry results. The CF RT used Epic MyChart messages to engage patients who needed education and to remind patients to use their device before all visits (virtual or face-to-face). The RT and APP also provided inperson teaching and troubleshooting in clinic, and an educational handout was created. Lastly, one-on-one educationwas provided over the phone on nonclinic days. Between November 2021 and January 2022, people with CF who had received a device and adult CF clinicians were surveyed for anonymous feedback on the program. Survey questions used a 5-point Likert scale to indicate agreement with the survey statement and were adapted from a previous home spirometry survey in lung transplant patients [1]. Result(s): Two hundred sixty-five patients (of 327 total adults with CF at UNC) have received a home spirometer through the CFF or another source;250 of these (94%) have received one-on-one education. All six adult CF clinicians completed the feedback survey. The results indicated a high level of satisfaction and a sense that spirometer use was clinically useful (Figure 1). Seventy-eight patients completed the feedback survey. Regarding the program's utility, respondents agreed or strongly agreed with the following statements: spirometer is easy to use (81%), received adequate training (88%), spirometer is accurate (78%), team is interested in my results (96%), makes me feel secure in detecting problems with my lungs (60%), helpful to share results on the provider dashboard (68%), and am overall satisfied with the home spirometry program (78%). When patients were asked about barriers to use of their home spirometer, 48 (62%) selected "none." The most commonly identified barriers (number of responses)were anxiety (5), too difficult to use (4), too time consuming (3), forgetfulness (3), and accuracy (2).(Figure Presented) Figure 1. Adult cystic fibrosis clinician survey results on home spirometry Conclusion(s):Wewere able to leverage thewidespread availability of home spirometers to create a successful home spirometry program within the UNC adult CF clinic. Our survey results indicate that clinicians and patients felt that home spirometry was useful and were highly satisfied with the program Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Development of the prism-coupler model for the design of a biosensor based on SPR technology for fast diagnostics

Biosensors are considered the tools of the future in medicine. With the pandemic caused by the new corona-virus (SARS-COV-2), the development of biosensors with fast diagnostics has become a public health need. Optical biosensors, in particular, have gained strength in the market due to their high sensitivity and detection capability. In this category, sensors based on surface plasmon resonance technology have gained prominence. In this work, a prism-coupler set using the Ktreschamnn configuration was designed and built. The prism-coupler smodel consists of an automated optical-mechanical system and a triangular prism. Thin films of 50 nm of gold and 5 nm of titanium were deposited on glass slices and coupled to a commercial prism (refractive index 1.50). The movement of the optical-mechanical system and the data acquisition are carried out by 2 microcontrollers that are configured using a Labview software. A simulation program using MatLab software was also developed in this work to determine the SPR curves at different refractive index solutions. At first, measurement in air was carried out obtaining a resonance angle in 35 degrees. Afterwards, sucrose solutions were measured using our system at concentrations of 10, 20, 30, 40 and 50%. Finally, experimental and simulated results were compared and analyzed in this work.

Boosting the detection performance of severe acute respiratory syndrome coronavirus 2 test through a sensitive optical biosensor with new superior antibody.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus emerged in late 2019 leading to the COVID-19 disease pandemic that triggered socioeconomic turmoil worldwide. A precise, prompt, and affordable diagnostic assay is essential for the detection of SARS-CoV-2 as well as its variants. Antibody against SARS-CoV-2 spike (S) protein was reported as a suitable strategy for therapy and diagnosis of COVID-19. We, therefore, developed a quick and precise phase-sensitive surface plasmon resonance (PS-SPR) biosensor integrated with a novel generated anti-S monoclonal antibody (S-mAb). Our results indicated that the newly generated S-mAb could detect the original SARS-CoV-2 strain along with its variants. In addition, a SARS-CoV-2 pseudovirus, which could be processed in BSL-2 facility was generated for evaluation of sensitivity and specificity of the assays including PS-SPR, homemade target-captured ELISA, spike rapid antigen test (SRAT), and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Experimentally, PS-SPR exerted high sensitivity to detect SARS-CoV-2 pseudovirus at 589 copies/ml, with 7-fold and 70-fold increase in sensitivity when compared with the two conventional immunoassays, including homemade target-captured ELISA (4 × 103 copies/ml) and SRAT (4 × 104 copies/ml), using the identical antibody. Moreover, the PS-SPR was applied in the measurement of mimic clinical samples containing the SARS-CoV-2 pseudovirus mixed with nasal mucosa. The detection limit of PS-SPR is calculated to be 1725 copies/ml, which has higher accuracy than homemade target-captured ELISA (4 × 104 copies/ml) and SRAT (4 × 105 copies/ml) and is comparable with qRT-PCR (1250 copies/ml). Finally, the ability of PS-SPR to detect SARS-CoV-2 in real clinical specimens was further demonstrated, and the assay time was less than 10 min. Taken together, our results indicate that this novel S-mAb integrated into PS-SPR biosensor demonstrates high sensitivity and is time-saving in SARS-CoV-2 virus detection. This study suggests that incorporation of a high specific recognizer in SPR biosensor is an alternative strategy that could be applied in developing other emerging or re-emerging pathogenic detection platforms.

QUANTIFICATION AND KINETIC PROFILING OF ANTI-RBD ANTIBODIES IN COVID-19 SERUM AND WHOLE BLOOD USING FO-SPR PLATFORM

We present fiber optic surface plasmon resonance (FO-SPR) label-free (LF) bioassays for quantification and kinetic profiling of complete antibody isotypes against the receptor binding domain (RBD) of SARS-CoV-2 spike protein. This was accomplished not only in serum but also for the first time directly in whole blood of COVID-19 convalescent patients. The LF bioassay was correlated with the traditional FO-SPR sandwich bioassay, the latter also benchmarked with ELISA. Compared to other serological tests, our approach is superior in: (1) information about kinetics, (2) rapid insight into the amount of all antibody isotypes and (3) exceptional compatibility with whole blood samples. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Equine Anti-SARS-CoV-2 Serum (ECIG) Binds to Mutated RBDs and N Proteins of Variants of Concern and Inhibits the Binding of RBDs to ACE-2 Receptor.

The COVID-19 pandemic caused by the severe acute syndrome virus 2 (SARS-CoV-2) has been around since November 2019. As of early June 2022, more than 527 million cases were diagnosed, with more than 6.0 million deaths due to this disease. Coronaviruses accumulate mutations and generate greater diversity through recombination when variants with different mutations infect the same host. Consequently, this virus is predisposed to constant and diverse mutations. The SARS-CoV-2 variants of concern/interest (VOCs/VOIs) such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28/P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) have quickly spread across the world. These VOCs and VOIs have accumulated mutations within the spike protein receptor-binding domain (RBD) which interacts with the angiotensin-2 converting enzyme (ACE-2) receptor, increasing cell entry and infection. The RBD region is the main target for neutralizing antibodies; however, other notable mutations have been reported to enhance COVID-19 infectivity and lethality. Considering the urgent need for alternative therapies against this virus, an anti-SARS-CoV-2 equine immunoglobulin F(ab')2, called ECIG, was developed by the Butantan Institute using the whole gamma-irradiated SARS-CoV-2 virus. Surface plasmon resonance experiments revealed that ECIG binds to wild-type and mutated RBD, S1+S2 domains, and nucleocapsid proteins of known VOCs, including Alpha, Gamma, Beta, Delta, Delta Plus, and Omicron. Additionally, it was observed that ECIG attenuates the binding of RBD (wild-type, Beta, and Omicron) to human ACE-2, suggesting that it could prevent viral entry into the host cell. Furthermore, the ability to concomitantly bind to the wild-type and mutated nucleocapsid protein likely enhances its neutralizing activity of SARS-CoV-2. We postulate that ECIG benefits COVID-19 patients by reducing the infectivity of the original virus and existing variants and may be effective against future ones. Impacting the course of the disease, mainly in the more vulnerable, reduces infection time and limits the appearance of new variants by new recombination.