Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection.
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ABSTRACT:
SUBMITTER: Elledge SK
PROVIDER: S-EPMC7444307 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Engineered immunoglobulin-G molecules (IgGs) are of wide interest for the development of detection elements in protein-based biosensors with clinical applications. The strategy usually employed for the de novo design of such engineered IgGs consists on merging fragments of the three-dimensional structure of a native IgG, which is immobilized on the biosensor surface, and of an antibody with an exquisite target specificity and affinity. In this work conventional and accelerated classical molecular dynamics (cMD and aMD, respectively) simulations have been used to propose two IgG-like antibodies for COVID-19 detection. More specifically, the crystal structure of the IgG1 B12 antibody, which inactivates the human immunodeficiency virus-1, has been merged with the structure of the antibody CR3022 Fab tightly bounded to SARS-CoV-2 receptor-binding domain (RBD) and the structure of the S309 antibody Fab fragment complexed with SARS-CoV-2 RBD. The two constructed antibodies, named IgG1-CR3022 and IgG1-S309, respectively, have been immobilized on a stable gold surface through a linker. Analyses of the influence of both the merging strategy and the substrate on the stability of the two constructs indicate that the IgG1-S309 antibody better preserves the neutralizing structure than the IgG1-CR3022 one. Overall, results indicate that the IgG1-S309 is appropriated for the generation of antibody based sensors for COVID-19 diagnosis.
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Project description:The spread of the COVID-19 pandemic around the world has revealed that it is urgently important to develop rapid and inexpensive assays for antibodies in general and anti-SARS-CoV-2 IgG antibody (anti-SARS-CoV-2 spike glycoprotein S1 antibody) in particular. Herein we report a method to detect the anti-SARS-CoV-2 spike antibody level by using Janus emulsions or Janus particles as biosensors. Janus emulsions are composed of two immiscible hydrocarbon and fluorocarbon oils. The hydrocarbon/water interfaces are functionalized with a secondary antibody of IgG protein and SARS-CoV-2 spike receptor binding domain (RBD), to produce two different Janus emulsions. Mixtures of these Janus droplets enable the detection of the anti-SARS-CoV-2 spike IgG antibody in an agglutination assay caused by the antibody's binding to both the secondary antibody of IgG antibody and SARS-CoV-2 spike protein RBD. Both qualitative optical images and quantitative fluorescence spectra are able to detect the level of anti-SARS-CoV-2 spike antibody at concentrations as low as 0.2 μg/mL in 2 h. The detection results of clinical human serum samples using this agglutination assay confirm that this method is applicable to clinical samples with good sensitivity and specificity. The reported method is generalizable and can be used to detect other analytes by attaching different biomolecular recognition elements to the surface of the Janus droplets.
| S-EPMC8231659 | biostudies-literature
Project description:The COVID-19 pandemic brings unprecedented crisis for public health and economics in the world. Detecting specific antibodies to SARS-CoV-2 is a powerful supplement for the diagnosis of COVID-19 and is important for epidemiological studies and vaccine validations. Herein, a rapid and quantitative detection method of anti-SARS-CoV-2 IgG antibody was built based on the optofluidic point-of-care testing fluorescence biosensor. Without complicated steps needed, the portable system is suitable for on-site sensitive determination of anti-SARS-CoV-2 IgG antibody in serum. Under the optimal conditions, the whole detection procedure is about 25 min with a detection limit of 12.5 ng/mL that can well meet the diagnostic requirements. The method was not obviously affected by IgM and serum matrix and demonstrated to have good stability and reliability in real sample analysis. Compared to ELISA test results, the proposed method exhibits several advantages including wider measurement range and easier operation. The method provides a universal platform for rapid and quantitative analysis of other related biomarkers, which is of significance for the prevention and control of COVID-19 pandemic.
| S-EPMC8363510 | biostudies-literature
Project description:SARS-CoV-2, the virus that causes COVID-19, has killed over 3 million people worldwide. Despite the urgency of the current pandemic, most available diagnostic methods for COVID-19 use RT-PCR to detect nucleic acid sequences specific to SARS-CoV-2. These tests are limited by their requirement of a large laboratory space, high reagent costs, multistep sample preparation, and the potential for cross-contamination. Moreover, results usually take hours to days to become available. Therefore, fast, reliable, inexpensive, and scalable point-of-care diagnostics are urgently needed. Here, we describe RAPID 1.0, a simple, handheld, and highly sensitive miniaturized biosensor modified with human receptor angiotensin-converting enzyme-2. RAPID 1.0 can detect SARS-CoV-2 using 10 μL of sample within 4 min through its increased resistance to charge transfer of a redox probe measured by electrochemical impedance spectroscopy. The sensitivity and specificity of RAPID for nasopharyngeal/oropharyngeal swab and saliva samples are 85.3% and 100% and 100% and 86.5%, respectively.
| S-EPMC8106877 | biostudies-literature
Project description:Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.
| S-EPMC8482318 | biostudies-literature
Project description:Rationale & objectiveA number of serologic tests for immunoglobulin G (IgG) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now commercially available, including multiple lateral flow immunoassays (LFIAs), which have the advantage of being inexpensive and easy to use, without the reliance on laboratory facilities. However, data on the development of humoral immunity to SARS-CoV-2 in patients with kidney disease is limited, and the utility of an LFIA to test for antibodies in these patients has not been assessed.Study designObservational study.Setting & participants60 patients (40 hemodialysis and 20 kidney transplant recipients) with SARS-CoV-2 infection confirmed by viral reverse transcriptase-polymerase chain reaction (RT-PCR) testing and 88 historic negative-control samples (collected before September 2019).TestA commercially available LFIA to test for SARS-CoV-2 IgG in patients with infection confirmed by viral RT-PCR testing.OutcomesSensitivity and specificity of the LFIA to detect SARS-CoV-2 IgG in dialysis patients and transplant recipients.Results56/58 (96.6%) patients (38/39 hemodialysis and 18/19 transplant recipients) tested positive for SARS-CoV-2 IgG. 5/7 (71.4%) patients who were negative on preliminary testing had detectable IgG when retested more than 21 days postdiagnosis. Median times to first and second tests after diagnosis were 17 (interquartile range, 15-20) and 35 (interquartile range, 30-39) days, respectively. Calculation of test characteristics gave sensitivity of 96.6% (95% CI, 88.3%-99.4%) and specificity of 97.7% (95% CI, 92.0-99.6%).LimitationsPossible exposure to other beta-coronaviruses that may cross-react with the antigen used in the LFIA cannot be excluded.ConclusionsSymptomatic dialysis patients and transplant recipients commonly develop an immune response against SARS-CoV-2 infection that can be detected using an LFIA. Used diligently, an LFIA could be used to help screen the dialysis populations or confirm exposure on a patient level, especially in facilities in which laboratory resources are limited.
| S-EPMC7834810 | biostudies-literature
Project description:The rapid detection of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is necessary in the ongoing pandemic. Antigen-specific point-of-care tests (POCT) may be useful for this purpose. Here, such a POCT (SARS-CoV-2 NADAL® COVID-19 Ag) was compared to a laboratory-developed triplex real-time polymerase chain reaction (RT-PCR) designed for the detection of viral nucleoprotein gene and two control targets. This RT-PCR served as a reference to investigate POCT sensitivity by re-testing upper respiratory tract (URT) samples (n = 124) exhibiting different SARS-CoV-2 loads in terms of RT-PCR threshold cycle (Ct) values. The optical intensities of the antigen bands were compared to the Ct values of the RT-PCR. The infectivity of various virus loads was estimated by inoculating Vero cells with URT samples (n = 64, Ct 17-34). POCT sensitivity varied from 100% (Ct < 25) to 73.1% (Ct ? 30); higher SARS-CoV-2 loads correlated with higher band intensities. All samples with a Ct > 30 were negative; among SARS-CoV-2 free samples (n = 10) no false-positives were detected. A head-to-head comparison with another POCT (Abbott, Panbio™ COVID-19 Ag Rapid Test) yielded similar results. Isolation of SARS-CoV-2 in cell-culture was successful up to a Ct value of 29. The POCT reliably detects high SARS-CoV-2 loads and rapidly identifies infectious individuals.
| S-EPMC7823488 | biostudies-literature