Project description:Early detection and identification of SARS-CoV-infected patients and actions to prevent transmission are absolutely critical to prevent another SARS outbreak. Antibodies that specifically recognize the SARS-CoV spike and nucleocapsid proteins may provide a rapid screening method to allow accurate identification and isolation of patients with the virus early in their infection. For this reason, we raised peptide-induced polyclonal antibodies against SARS-CoV spike protein and polyclonal antibodies against SARS-CoV nucleocapsid protein using 6x His nucleocapsid recombinant protein. Western blot analysis and immunofluorescent staining showed that these antibodies specifically recognized SARS-CoV.
Project description:Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NP NTD ) from SARS-CoV-2 using small angle X-ray scattering (SAXS). Our solution-based results distinguished the mAbs' flexibility and how this flexibility impacts the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NP NTD , we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the Fabs is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich ELISA, we show the rigid mAb-pairings with linear polymerization led to increased NP NTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices (LFA), key for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.
Project description:Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs' flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.
Project description:The goal of this work was to develop recombinantly expressed variable domains derived from camelid heavy-chain antibodies known as single-domain antibodies (sdAbs) directed against the SARS-CoV-2 nucleocapsid protein for incorporation into detection assays. To achieve this, a llama was immunized using a recombinant SARS-CoV-2 nucleocapsid protein and an immune phage-display library of variable domains was developed. The sdAbs selected from this library segregated into five distinct sequence families. Three of these families bind to unique epitopes with high affinity, low nM to sub-nM KD, as determined by surface plasmon resonance. To further enhance the utility of these sdAbs for the detection of nucleocapsid protein, homobivalent and heterobivalent genetic fusion constructs of the three high-affinity sdAbs were prepared. The effectiveness of the sdAbs for the detection of nucleocapsid protein was evaluated using MagPlex fluid array assays, a multiplexed immunoassay on color-coded magnetic microspheres. Using the optimal bivalent pair, one immobilized on the microsphere and the other serving as the biotinylated recognition reagent, a detection limit as low as 50 pg/mL of recombinant nucleocapsid and of killed virus down to 1.28 × 103 pfu/mL was achieved. The sdAbs described here represent immune reagents that can be tailored to be optimized for a number of detection platforms and may one day aid in the detection of SARS-CoV-2 to assist in controlling the current pandemic.
Project description:Since the COVID-19 disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) was declared a pandemic, it has spread rapidly, causing one of the most serious outbreaks in the last century. Reliable and rapid diagnostic tests for COVID-19 are crucial to control and manage the outbreak. Here, a label-free square wave voltammetry-based biosensing platform for the detection of SARS-CoV-2 in nasopharyngeal samples is reported. The sensor was constructed on screen-printed carbon electrodes coated with gold nanoparticles. The electrodes were functionalized using 11-mercaptoundecanoic acid (MUA) which was used for the immobilization of an antibody against SARS-CoV-2 nucleocapsid protein (N protein). The binding of the immunosensor with the N protein caused a change in the electrochemical signal. The detection was realised by measuring the change in reduction peak current of a redox couple using square wave voltammetry at 0.04 V versus Ag ref. electrode on the immunosensor upon binding with the N protein. The electrochemical immunosensor showed high sensitivity with a linear range from 1.0 pg.mL-1 to 100 ng.mL-1 and a limit of detection of 0.4 pg.mL-1 for the N protein in PBS buffer pH 7.4. Moreover, the immunosensor did not exhibit significant response with other viruses such as HCoV, MERS-CoV, Flu A and Flu B, indicating the high selectivity of the sensor for SARS-CoV-2. However, cross reactivity of the biosensor with SARS-CoV is indicated, which gives ability of the sensor to detect both SARS-CoV and SARS-CoV-2. The biosensor was successfully applied to detect the SARS-CoV-2 virus in clinical samples showing good correlation between the biosensor response and the RT-PCR cycle threshold values. We believe that the capability of miniaturization, low-cost and fast response of the proposed label-free electrochemical immunosensor will facilitate the point-of-care diagnosis of COVID 19 and help prevent further spread of infection.
Project description:Profiling antibodies to SARS-CoV-2 can help to assess potential immune response after COVID-19 disease. Luciferase IP system (LIPS) assay is a sensitive method for quantitative detection of antibodies to antigens in their native conformation. We here describe LIPS to detect antibody responses to SARS-CoV-2 spike (S) and nucleocapsid (N) proteins in COVID-19 patients. The antibodies targeted both S and N fragments and gave a high assay sensitivity by identifying 26 out of 26 COVID-19 patients with N antigen or with three protein fragments when combined into a single reaction. The assay correlated well with ELISA method and was specific to COVID-19 as we saw no reactivity among uninfected healthy controls. Our results show that LIPS is a rapid and measurable method to screen antibody responses against SARS-CoV-2 antigens.
Project description:Rapid, mass diagnosis of the coronavirus disease 2019 (COVID-19) is critical to stop the ongoing infection spread. The two standard screening methods to confirm the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are polymerase chain reaction (PCR), through the RNA of the virus, and serology by detecting antibodies produced as a response to the viral infection. However, given the detection complexity, cost and relatively long analysis times of these techniques, novel technologies are urgently needed. Here, we report an aptamer-based biosensor developed on a screen-printed carbon electrode platform for rapid, sensitive, and user-friendly detection of SARS-CoV-2. The aptasensor relies on an aptamer targeting the receptor-binding domain (RBD) in the spike protein (S-protein) of the SARS-CoV-2. The aptamer immobilization on gold nanoparticles, and the presence of S-protein in the aptamer-target complex, investigated for the first time by photo-induced force microscopy mapping between 770 and 1910 cm-1 of the electromagnetic spectrum, revealed abundant S-protein homogeneously distributed on the sensing probe. The detection of SARS-CoV-2 S-protein was achieved by electrochemical impedance spectroscopy after 40 min incubation with several analyte concentrations, yielding a limit of detection of 1.30 pM (66 pg/mL). Moreover, the aptasensor was successfully applied for the detection of a SARS-CoV-2 pseudovirus, thus suggesting it is a promising tool for the diagnosis of COVID-19.
Project description:The availability of sensors able to rapidly detect SARS-CoV-2 directly in biological fluids in a single step would allow performing massive diagnostic testing to track in real time and contain the spread of COVID-19. Motivated by this, here, we developed an electrochemical aptamer-based (EAB) sensor able to achieve the rapid, reagentless, and quantitative measurement of the SARS-CoV-2 spike (S) protein. First, we demonstrated the ability of the selected aptamer to undergo a binding-induced conformational change in the presence of its target using fluorescence spectroscopy. Then, we engineered the aptamer to work as a bioreceptor in the EAB platform and we demonstrated its sensitivity and specificity. Finally, to demonstrate the clinical potential of the sensor, we tested it directly in biological fluids (serum and artificial saliva), achieving the rapid (minutes) and single-step detection of the S protein in its clinical range.
Project description:The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) emerged at the end of 2019, resulting in the ongoing COVID-19 pandemic. The high transmissibility of the virus and the substantial number of asymptomatic individuals have led to an exponential rise in infections worldwide, urgently requiring global containment strategies. Reverse transcription-polymerase chain reaction is the gold standard for the detection of SARS-CoV-2 infections. Antigen tests, targeting the spike (S) or nucleocapsid (N) viral proteins, are considered as complementary tools. Despite their shortcomings in terms of sensitivity and specificity, antigen tests could be deployed for the detection of potentially contagious individuals with high viral loads. In this work, we sought to develop a sandwich aptamer-based assay for the detection of the S protein of SARS-CoV-2. A detailed study on the binding properties of aptamers to the receptor-binding domain of the S protein in search of aptamer pairs forming a sandwich is presented. Screening of aptamer pairs and optimization of assay conditions led to the development of a laboratory-based sandwich assay able to detect 21 ng/mL (270 pM) of the protein with negligible cross-reactivity with the other known human coronaviruses. The detection of 375 pg of the protein in viral transport medium demonstrates the compatibility of the assay with clinical specimens. Finally, successful detection of the S antigen in nasopharyngeal swab samples collected from suspected patients further establishes the suitability of the assay for screening purposes as a complementary tool to assist in the control of the pandemic.