Due to urgency and demand, numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoassays are rapidly being developed and placed on the market with limited validation on clinical samples. Thorough validation of serological tests are required to facilitate their use in the accurate diagnosis of SARS-CoV-2 infection, confirmation of molecular results, contact tracing, and epidemiological studies. This study evaluated the sensitivity and specificity of nine commercially available serological tests. These included three enzyme-linked immunosorbent assays (ELISAs) and six point-of-care (POC) lateral flow tests. The assays were validated using serum samples from: i) SARS-CoV-2 PCR-positive patients with a documented first day of disease; ii) archived sera obtained from healthy individuals before the emergence of SARS-CoV-2 in China;iii) sera from patients with acute viral respiratory tract infections caused by other coronaviruses or noncoronaviruses; and iv) sera from patients positive for dengue virus, cytomegalovirus and Epstein Barr virus.The results showed 100% specificity for the Wantai SARS-CoV-2 Total Antibody ELISA, 93% for the Euroimmun IgA ELISA, and 96% for the Euroimmun IgG ELISA with sensitivities of 90%, 90%, and 65%, respectively. The overall performance of the POC tests according to manufacturer were in the rank order of AutoBio Diagnostics > Dynamiker Biotechnology = CTK Biotech > Artron Laboratories > Acro Biotech ≥ Hangzhou Alltest Biotech. Overall, these findings will facilitate selection of serological assays for the detection SARS-CoV-2-specific antibodies towards diagnosis as well as sero-epidemiological and vaccine development studies.
Severe acute respiratory syndrome coronavirus 2 has caused a pandemic in humans. Farmed mink ( Neovison vison ) are also susceptible. In Denmark, this virus has spread rapidly among farmed mink, resulting in some respiratory disease. Full-length virus genome sequencing revealed novel virus variants in mink. These variants subsequently appeared within the local human community.
SARS-CoV-2 infection is the cause of COVID-19 in humans. In April 2020, SARS-CoV-2 infection in farmed mink (Neovision vision) occurred in the Netherlands. The first outbreaks in Denmark were detected in June 2020 in three farms. A steep increase in the number of infected farms occurred from September and onwards. Here, we describe prevalence data collected from 215 infected mink farms to characterize spread and impact of disease in infected farms. In one third of the farms, no clinical signs were observed. In farms with clinical signs, decreased feed intake, increased mortality and respiratory symptoms were most frequently observed, during a limited time period (median of 11 days). In 65% and 69% of farms, virus and sero-conversion, respectively, were detected in 100% of sampled animals at the first sampling. SARS-CoV-2 was detected, at low levels, in air samples collected close to the mink, on mink fur, on flies, on the foot of a seagull, and in gutter water, but not in feed. Some dogs and cats from infected farms tested positive for the virus. Chickens, rabbits, and horses sampled on a few farms, and wildlife sampled in the vicinity of the infected farms did not test positive for SARS-CoV-2. Thus, mink are highly susceptible to infection by SARS-CoV-2, but routes of transmission between farms, other than by direct human contact, are unclear.
Globally, the COVID-19 pandemic has had extreme consequences for the healthcare system and has led to calls for diagnostic tools to monitor and understand the transmission, pathogenesis, and epidemiology, as well as to evaluate future vaccination strategies. In this study, we have developed novel, to our knowledge, flexible ELISA-based assays for specific detection of human SARS-CoV-2 Abs against the receptor-binding domain, including an Ag sandwich ELISA relevant for large population screening and three isotype-specific assays for in-depth diagnostics. Their performance was evaluated in a cohort of 350 convalescent participants with previous COVID-19 infection, ranging from asymptomatic to critical cases. We mapped the Ab responses to different areas on protein N and S and showed that the IgM, A, and G Ab responses against receptor-binding domain are significantly correlated to the disease severity. These assays and the data generated from them are highly relevant for diagnostics and prognostics and contribute to the understanding of long-term COVID-19 immunity.
Serological SARS-CoV-2 assays are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (total-Ab) and immunoglobulin (Ig) G and M against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays. This study was a Danish national collaboration and evaluated fifteen commercial and one in-house anti-SARS-CoV-2 assays in sixteen laboratories. Sensitivity was evaluated using 150 samples from individuals with asymptomatic, mild or moderate COVID-19; nonhospitalized or hospitalized, confirmed by nucleic acid amplification tests (NAAT), collected 13-73 days either from symptom onset or from positive NAAT (patients without symptoms). Specificity and cross reactivity were evaluated in samples collected prior to the SARS-CoV-2 epidemic from >586 blood donors and patients with autoimmune diseases, cytomegalovirus or Epstein-Barr virus infections and acute viral infections. A specificity of ≥99% was achieved by all total-Ab and IgG assays except one, Diasorin/LiaisonXL-IgG (97.2%). Sensitivities in descending order were: Wantai/ELISA total-Ab (96.7%), CUH-NOVO/in-house ELISA total-Ab (96.0%), Ortho/Vitros total-Ab (95.3%), YHLO/iFlash-IgG (94.0%), Ortho/Vitros-IgG (93.3%), Siemens/Atellica total-Ab (93.2%), Roche/Elecsys total-Ab (92.7%), Abbott/Architect-IgG (90.0%), Abbott/Alinity-IgG (median 88.0%), Diasorin/LiaisonXL-IgG (median 84.6%), Siemens/Vista total-Ab (81.0%), Euroimmun/ELISA-IgG (78.0%), and Snibe/Maglumi-IgG (median 78.0%). However, confidence intervals overlapped for several assays. The IgM results were variable, with the Wantai/ELISA-IgM showing the highest sensitivity (82.7%) and specificity (99%). The rate of seropositivity increased with time from symptom onset and symptom severity.
Enzyme-linked immunosorbent assay (ELISA) is a validated and sensitive method for detection of human autoantibodies, but may have problems with specificity. Non-specific binding is a well-known problem often observed in tests for autoantibodies, when sera are incubated on plastic surfaces, e.g. an ELISA plate. To understand the mechanisms underlying non-specific immunoglobulin deposition, we here analyse the phenomenon in detail and we propose means of reducing false positive test results caused by non-specific binding. The level of non-specific binding, in sera with suspected autoreactivity, was analysed in non-coated and autoantigen-coated ELISA wells and 4-32% of sera showed a high level of non-specific binding depending on the assay conditions and serum properties. Non-specifically binding sera were found to contain increased concentrations of IgG and other inflammatory mediators. Moreover, non-specific binding could be induced in serum by increasing the concentration of IgG and incubating the serum at 40 °C. This suggests that non-specific binding immunoglobulins can be formed during inflammation with high immunoglobulin levels and elevated temperature. We show that the level of non-specific binding correlates with the IgG concentration and therefore propose that non-specific binding may be interpreted as an informative finding indicative of elevated IgG and inflammation.
The chaperone calreticulin is a highly conserved eukaryotic protein mainly located in the endoplasmic reticulum. It contains a free cysteine SH group but does not form disulfide-bridged dimers under physiological conditions, indicating that the SH group may not be fully accessible in the native protein. Using PAGE, urea gradient gel electrophoresis, capillary electrophoresis and MS, we show that dimerization through the SH group can be induced by lowering the pH to 5-6, heating, or under conditions that favour partial unfolding such as urea concentrations above 2.6 M or SDS concentrations above 0.025%. Moreover, we show that calreticulin also has the ability to self-oligomerize through noncovalent interactions at urea concentrations above 2.6 M at pH below 4.6 or above pH 10, at temperatures above 40°C, or in the presence of high concentrations of organic solvents (25%), conditions that favour partial unfolding or an intramolecular local conformational change that allows oligomerization, resulting in a heterogeneous mixture of oligomers consisting of up to 10 calreticulin monomers. The oligomeric calreticulin was very stable, but oligomerization was partially reversed by addition of 8 M urea or 1% SDS, and heat-induced oligomerization could be inhibited by 8 M urea or 1% SDS when present during heating. Comparison of the binding properties of monomeric and oligomeric calreticulin in solid-phase assays showed increased binding to peptides and denatured proteins when calreticulin was oligomerized. Thus, calreticulin shares the ability to self-oligomerize with other important chaperones such as GRP94 and HSP90, a property possibly associated with their chaperone activity.
Seroprevalence studies have proven an important tool to monitor the progression of the coronavirus disease 2019 (COVID-19) pandemic. We present results of consecutive population-based seroprevalence surveys performed in Denmark in 2020. In spring, late summer and autumn/winter of 2020, invitation letters including a questionnaire covering symptoms were sent to representative samples of the population above 12 years and to parents of children below 18 years in the sample. Blood samples were analysed for total Ig and seroprevalence estimates per population segment were calculated and compared to other surveillance parameters. Based on 34 081 participants (participation rate 33%), seroprevalence estimates increased from 1.2% (95%CI: 0.3–1.9%) in May to 4.1% (95%CI: 3.1–4.9%) in December 2020. Seroprevalence estimates were roughly three times higher in those aged 12–29 years compared to 65 + and higher in metropolitan municipalities. By December 2020, 1.5% of the population had tested positive by RT-PCR. Infected individuals in older age groups were hospitalised several fold more often than in younger. Amongst seropositives, loss of taste/smell were the more specific symptoms, 32–56% did not report any symptoms. In more than half of seroconverted families, we did not see evidence of transmission between generations. Seroprevalence increased during 2020; adolescents were primarily infected in the autumn/winter. Denmark has a high per capita test rate; roughly one undiagnosed infection of SARS-CoV-2 were estimated to occur for each diagnosed case. Approximately half were asymptomatically infected. The epidemic appears to have progressed relatively modestly during 2020 in Denmark.
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