Abstract:The pandemic of COVID‐19 has caused enormous fatalities worldwide. Serological assays are important for detection of asymptomatic or mild cases of COVID‐19, and sero‐prevalence and vaccine efficacy studies. Here, we evaluated and compared the performance of seven commercially available enzyme‐linked immunosorbent assay (ELISA)s for detection of anti‐severe acute respiratory syndrome corona virus 2 (SARS‐CoV‐2) immunoglobulin G (IgG). The ELISAs were evaluated with a characterized panel of 100 serum samples fro… Show more
“…However, there are many limitations to using PCR in diagnosis, including low sensitivity and the complex processes that could not meet the urgency of the early diagnosis of the vast numbers of suspected cases, as well as patients' discomfort during nasopharyngeal swabs [18]. Many commercial kits are available for antibodies detection but with uncertain performance [19]. However, earlier diagnosis by antigen-based detection system in the acute phase of the disease is much better for accurate results.…”
The development of sensitive, non-invasive tests for the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens is imperative, and it is still challenging to manage the extent of infection throughout the population. Here, we designed and optimized a sandwich enzyme-linked immunosorbent assay (ELISA) protocol for SARS-CoV-2 S1 antigen detection in saliva. Both saliva samples and nasopharyngeal swabs were collected from 220 real-time quantitative polymerase chain reaction (RT-qPCR)-confirmed positive and negative cases. S1 protein receptor-binding domain (RBD) nanobodies were efficiently conjugated with 40 nm gold nanoparticles (AuNPs) and employed as antigen detection probes in the developed system, while recombinant S1 monoclonal antibodies (S1mAbs) were employed as antigen capture probes. After checkerboard assays and system optimization, the clinical samples were tested. In saliva, the developed ELISA system showed the highest sensitivity (93.3) for samples with cycle threshold (Ct) values ≤ 30; interestingly, high sensitivity (87.5 and 86%) was also achieved for samples with Ct values ≤35 and ≤40, respectively, compared with 90, 80 and 88% sensitivity rates for nasopharyngeal swabs with the same categorized Ct values. However, the specificity was 100%, and no cross-reactions were detected with Middle East respiratory syndrome coronavirus (MERS-CoV) or SARS-CoV antigens. These results reveal that our protocol could be established as an efficient and sensitive, non-invasive diagnostic tool for the early detection of SARS-CoV-2 infection using easily collectable saliva samples.
“…However, there are many limitations to using PCR in diagnosis, including low sensitivity and the complex processes that could not meet the urgency of the early diagnosis of the vast numbers of suspected cases, as well as patients' discomfort during nasopharyngeal swabs [18]. Many commercial kits are available for antibodies detection but with uncertain performance [19]. However, earlier diagnosis by antigen-based detection system in the acute phase of the disease is much better for accurate results.…”
The development of sensitive, non-invasive tests for the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens is imperative, and it is still challenging to manage the extent of infection throughout the population. Here, we designed and optimized a sandwich enzyme-linked immunosorbent assay (ELISA) protocol for SARS-CoV-2 S1 antigen detection in saliva. Both saliva samples and nasopharyngeal swabs were collected from 220 real-time quantitative polymerase chain reaction (RT-qPCR)-confirmed positive and negative cases. S1 protein receptor-binding domain (RBD) nanobodies were efficiently conjugated with 40 nm gold nanoparticles (AuNPs) and employed as antigen detection probes in the developed system, while recombinant S1 monoclonal antibodies (S1mAbs) were employed as antigen capture probes. After checkerboard assays and system optimization, the clinical samples were tested. In saliva, the developed ELISA system showed the highest sensitivity (93.3) for samples with cycle threshold (Ct) values ≤ 30; interestingly, high sensitivity (87.5 and 86%) was also achieved for samples with Ct values ≤35 and ≤40, respectively, compared with 90, 80 and 88% sensitivity rates for nasopharyngeal swabs with the same categorized Ct values. However, the specificity was 100%, and no cross-reactions were detected with Middle East respiratory syndrome coronavirus (MERS-CoV) or SARS-CoV antigens. These results reveal that our protocol could be established as an efficient and sensitive, non-invasive diagnostic tool for the early detection of SARS-CoV-2 infection using easily collectable saliva samples.
“…Enhancing the Sensitivity of Commercial LFA Strips through Hydrogel Modification. Compared to genetic material-based molecular diagnosis (e.g., quantitative reverse transcription-polymerase chain reaction (qRT-PCR)) 39 or antigen/antibody-based immunoassays (e.g., enzyme-linked immunosorbent assays (ELISA)), 40 which require skilled technicians, specific equipment, sophisticated operation, and strict environmental conditions for reagent transportation and storage, LFA is a more convenient and widely used screening tool for various pathogens under nonlaboratory scenarios owing to its low cost and simple operation. Many strategies using the LFA format have been successfully developed for COVID-19 diagnosis in the pandemic situation; the recently presented test strips even could achieve comparable sensitivity with RT-PCR technology.…”
Section: ■ Results and Discussionmentioning
confidence: 99%
“…Considering only the materials required for the CL, the cost of an HCL containing 3% agarose, 16% gelatin, or 1.5% κ-carrageenan was 99.68, 99.55, and 99.38% lower, respectively, compared to an IgG (Table S2). Furthermore, the traditional approach of obtaining antibody pairs involves the laborious process of preparing monoclonal antibodies and subsequent labeling, followed by extensive screening of potential capture antibody pairs using sandwich ELISA to identify the best combinations . This method is time-consuming and does not facilitate the rapid development of antigen detection methods .…”
“…Standard immunological assays on the contrary are easy to perform, easy to upscale, less time consuming and does not require BSL3 facilities. Several correlation studies have been performed assessing the applicability of other immunological assays such as ELISA for the use in functional antibody screening [28][29][30][31]. Some report a good correlation between ELISA and neutralization assays [28,30], while others report poor agreement, leaving other serological assays less fit-for-purpose for use in convalescent plasma therapy and other antibody functionality studies [29].…”
Virus neutralization assays provide a means to quantitate functional antibody responses that block virus infection. These assays are instrumental in defining vaccine and therapeutic antibody potency, immune evasion by viral variants, and post-infection immunity. Here we describe the development, optimization and evaluation of a live virus microneutralization assay specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this assay, SARS-CoV-2 clinical isolates are pre-incubated with serial diluted antibody and added to Vero E6 cells. Replicating virus is quantitated by enzyme-linked immunosorbent assay (ELISA) targeting the SARS-CoV-2 nucleocapsid protein and the standardized 50% virus inhibition titer calculated. We evaluated critical test parameters that include virus titration, assay linearity, number of cells, viral dose, incubation period post-inoculation, and normalization methods. Virus titration at 96 hours was determined optimal to account for different growth kinetics of clinical isolates. Nucleocapsid protein levels directly correlated with virus inoculum, with the strongest correlation at 24 hours post-inoculation. Variance was minimized by infecting a cell monolayer, rather than a cell suspension. Neutralization titers modestly decreased with increasing numbers of Vero E6 cells and virus amount. Application of two different normalization models effectively reduced the intermediate precision coefficient of variance to <16.5%. The SARS-CoV-2 microneutralization assay described and evaluated here is based on the influenza virus microneutralization assay described by WHO, and are proposed as a standard assay for comparing neutralization investigations.
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