BACKGROUND. In the COVID-19 pandemic, highly selective serological testing is essential to define exposure to SARS-CoV-2 virus. Many tests have been developed, yet with variable speed to first result, and of unknown quality, particularly when considering the prediction of neutralizing capacity. OBJECTIVES/METHODS. The LIAISON® SARS-CoV-2 S1/S2 IgG assay was designed to measure antibodies against the SARS-CoV-2 native S1/S2 proteins in a standardized automated chemiluminescent assay. Clinical and analytical performance of the test were validated in an observational study using residual samples (>1500) with positive or negative COVID-19 diagnosis. RESULTS. The LIAISON® SARS-CoV-2 S1/S2 IgG assay proved to be highly selective and specific, and offers semiquantitative measures of serum or plasma levels of anti-S1/S2 IgG with neutralizing activity. The assay's diagnostic sensitivity was 91.3% and 95.7% at >5 or ≥15 days from diagnosis, respectively, and 100% when assessed against a neutralizing assay. The assay's specificity ranged between 97% and 98.5%. The average imprecision of the assay was <5 % coefficient of variation. Assay performance at 2 different cut-offs was evaluated to optimize predictive values. CONCLUSIONS. The automated LIAISON® SARS-CoV-2 S1/S2 IgG assay brings efficient, sensitive, specific, and precise serological testing to the laboratory, with the capacity to test large amounts of samples per day: first results are available within 35 minutes with a throughput of 170 tests/hour. The semiquantitative results provided by the test also associate with the presence of neutralizing antibodies, and may provide a useful tool for the large scale screening of convalescent plasma for safe therapeutic use.
Escherichia coli both the complete core gene of hepatitis B virus and a truncated version of it, leading to the synthesis of high levels of a core-antigen-equivalent polypeptide (r-p22) and of an e-antigen-equivalent polypeptide (r-p16), respectively. We then compared the structural and antigenic properties of the two polypeptides, as well as their ability to bind viral nucleic acids. r-p16 was found to self-assemble into capsid-like particles that appeared similar, when observed under the electron microscope, to those formed by r-p22. In r-p16 particles, disulfide bonds linked the truncated polypeptides in dimers, assembled in the particle by noncovalent interactions. In r-p22 capsids, further disulfide bonds, conceivably involving the carboxy-terminal cysteines of r-p22 polypeptides, joined the dimers together, converting the structure into a covalently closed lattice. The protamine-like domain was at least partly exposed on the surface of r-p22 particles, since it was accessible to selective proteolysis. Finally, r-p22, but not r-p16, was shown to bind native and denatured DNA as well as RNA. Taken together, these results suggest that the protamine-like domain in core polypeptides is a nucleic acid-binding domain and is dispensable for the correct folding and assembly of amino-terminal and central regions.
Objectives COVID-19 has brought about tests from many manufacturers. While molecular and rapid antigen tests are targeted for early diagnosis, immunoassays have a larger role in epidemiological studies, understanding longitudinal immunity, and in vaccine development and response. Methods The performance of the LIAISON® SARS-CoV-2 TrimericS IgG assay was evaluated against the Beckman ACCESS SARS-CoV-2 IgG assay in New Mexico, and against the Siemens ADVIA Centaur COV2G assay in New York. Discordant samples were parsed using a microneutralization assay. Results A SARS-CoV-2 antibody positivity rate of 23.8% was observed in the samples tested in New York (September 2020), while in the same month the positivity rate was 1.5% in New Mexico. Positive and negative agreement were 67.6% (95% CI 49.5–82.6%) and 99.8% (95% CI 99.5–99.9%), respectively, with the Beckman test, and 98.0% (95% CI 95.7–99.3%) and 94.8% (95% CI 93.4–96.0%), respectively, with the Siemens test. Receiver operating characteristic analysis for the detection of SARS-CoV-2 antibodies discloses an AUC, area under the curve, of 0.996 (95% CI 0.992–0.999) for the LIAISON® SARS-CoV-2 TrimericS IgG assay. The criterion associated to the Youden Index was determined to be >12.9 kAU/L with a sensitivity of 99.44% and a specificity of 99.82%. Conclusions The LIAISON® SARS-CoV-2 TrimericS IgG assay is highly sensitive and specific. The balance of these parameters, without emphasis on high specificity alone, is particularly important when applied to high prevalence populations, where a highly sensitive assay will result in reporting a lower number of false negative subjects.
word count: 246 20 Text word count: 3188 21 22 4 ABSTRACT 23BACKGROUND. In the Covid-19 pandemic, highly selective serological testing is 24 essential to define exposure to SARS-CoV-2 virus. Many tests have been developed, 25 yet with variable speed to first result, and of unknown quality, particularly when 26 considering the prediction of neutralizing capacity. 27 OBJECTIVES/METHODS. The LIAISON ® SARS-CoV-2 S1/S2 IgG assay was designed 28 to measure antibodies against the SARS-CoV-2 native S1/S2 proteins in a standardized 29 automated chemiluminescent assay. Clinical and analytical performance of the test 30 were validated in an observational study using residual samples (>1500) with positive or 31 negative Covid-19 diagnosis. 32RESULTS. The LIAISON ® SARS-CoV-2 S1/S2 IgG assay proved highly selective and 33 specific, and offers semiquantitative measures of serum or plasma levels of anti-S1/S2 34 IgG with neutralizing activity. The diagnostic sensitivity was 91.3% and 95.7% at >5 or 35 ≥15 days from diagnosis respectively, and 100% when assessed against a neutralizing 36 assay. The specificity ranged between 97% and 98.5%. The average imprecision of the 37 assay was <5 % coefficient of variation. Assay performance at 2 different cut-offs was 38 evaluated to optimize predictive values in settings with different % disease prevalence. 39 CONCLUSIONS. The automated LIAISON ® SARS-CoV-2 S1/S2 IgG assay brings 40 efficient, sensitive, specific, and precise serological testing to the laboratory, with the 41 capacity to test large amounts of samples per day: first results are available within 35 42 minutes with a throughput of 170 tests/hour. The test also provides a semiquantitative 43 measure to identify samples with neutralizing antibodies, useful also for a large scale 44 screening of convalescent plasma for safe therapeutic use. 45 5 IMPORTANCE 46With the worldwide advance of the COVID-19 pandemic, efficient, reliable and 47 accessible diagnostic tools are needed to support public health officials and healthcare 48 providers in their efforts to deliver optimal medical care, and articulate sound 49 demographic policy. DiaSorin has developed an automated serology based assay for 50 the measurement of IgG specific to SARS CoV-2 Spike protein, and tested its clinical 51 performance in collaboration with Italian health care professionals who provided access 52 to large numbers of samples from infected and non-infected individuals. The assay 53 delivers excellent sensitivity and specificity, and is able to identify samples with high 54 levels of neutralizing antibodies. This will provide guidance in assessing the true 55 immune status of subjects, as well as meeting the pressing need to screen donors for 56 high titer convalescent sera for subsequent therapeutic and prophylactic use. 57 58 SARS-CoV-2, the virus responsible for the Covid-19 pandemic, has spread at an 59 alarming rate since the first case tracked back to mid-November of 2019 in Wuhan 60 China (1). Contraction and subsequent transmission accrue...
SARS-CoV-2 vaccination has proven effective in inducing an immune response in healthy individuals and is progressively us allowing to overcome the pandemic. Recent evidence has shown that response to vaccination in some vulnerable patients may be diminished, and it has been proposed a booster dose. We tested the kinetic of development of serum antibodies to the SARS-CoV-2 Spike protein, their neutralizing capacity, the CD4 and CD8 IFN-γ T-cell response in 328 subjects, including 131 immunocompromised individuals (cancer, rheumatologic, and hemodialysis patients), 160 health-care workers (HCW) and 37 subjects older than 75 yr, after vaccination with two or three doses of mRNA vaccines. We stratified the patients according to the type of treatment. We found that immunocompromised patients, depending on the type of treatment, poorly respond to SARS-CoV-2 mRNA vaccines. However, an additional booster dose of vaccine induced a good immune response in almost all of the patients except those receiving anti-CD20 antibody. Similarly to HCW, previously infected and vaccinated immunocompromised individuals demonstrate a stronger SARS-CoV-2–specific immune response than those who are vaccinated without prior infection.
ObjectivesVitamin D deficiency and hyperparathyroidism are common in patients with heart failure (HF). There is a growing body of evidence supporting the role of vitamin D and parathyroid hormone (PTH) in cardiac remodeling and worsening of HF. Lack of reliable automated testing of 1,25-dihydroxyvitamin D (1,25(OH)2D), the biologically active metabolite of vitamin D, has limited its contribution to the prognostic assessment of HF. Here, the association of 1,25(OH)2D and PTH(1–84) levels was evaluated for prediction of cardiovascular death in chronic HF patients.MethodsWe conducted a single center prospective cohort including 170 chronic HF patients (females n = 36; males n = 134; NYHA II-IV; mean age: 67 years; etiology: ischemic n = 119, dilated cardiomyopathy n = 51; mean LVEF: 23%). The primary outcome was cardiovascular death.ResultsSerum levels of 1,25(OH)2D decreased markedly with increased HF severity. Medians were 33.3 pg/mL for NYHA-II patients, 23.4 pg/mL for NYHA-III, and 14.0 pg/mL for NYHA-IV patients (p<0.001). Most patients had levels of 25(OH)D below 30ng/mL, and stratification by NYHA functional class did not show significant differences (p = 0.249). The 1,25(OH)2D to PTH(1–84) ratio and the (1,25(OH)2D)2 to PTH(1–84) ratio were found to be the most significantly related to HF severity. After a median follow-up of 4.1 years, 106 out of 170 patients reached the primary endpoint. Cox proportional hazard modeling revealed 1,25(OH)2D and the 1,25(OH)2D to PTH(1–84) ratios to be strongly predictive of outcomes.Conclusions1,25(OH)2D and its ratios to PTH(1–84) strongly and independently predict cardiovascular mortality in chronic HF.
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