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PurposeSARS-CoV-2 serology testing is key for assessing seroprevalence and antibody response post-vaccination in immunocompromised patients. Evaluation of current SARS-CoV-2 serological assays have been performed on samples from severe COVID-19 hospitalised patients. However, robust assay development requires assessment in asymptomatic and non-hospitalised individuals to determine if serological assays are sensitive to detect waning and mild antibody responses. Our study evaluated the performance characteristics between two high-throughput SARS-CoV-2 IgG nucleocapsid assays (Abbott and Roche) and The binding site (TBS) Anti-Spike IgG/A/M ELISA kit in healthcare workers.Methods236 samples were collected from Portsmouth Hospital University NHS Trust (PHU) and The Dudley Group NHS Trust and analysed for SARS-CoV-2 serology. We derived concordance, agreement and assay performance as well as using receiver operating characteristic (ROC) curves to redefine the assay threshold of the Abbott assay.ResultsResult concordance between the Abbott and TBS was 66%. Discrepant samples were analysed using the Roche assay which showed 100% agreement with the TBS assay. In samples analysed >58 days post-PCR, the sensitivity of Abbott and Roche was 100%. In samples analysed >100 days post-PCR the sensitivity of the Abbott assay dropped to 77.2% but remained at 100% for the Roche assay. A redefined Abbott threshold of 0.64 increased the sensitivity to 90% giving results similar to Roche and TBS assaysConclusionThis study demonstrated Abbott assay had a lower sensitivity in comparison to TBS and Roche. Furthermore, TBS can be implemented as a viable alternative for SARS-CoV-2 serology testing where high-throughput assays are not available on site.Trial registration number and date of registrationNot applicable.Trial registration number, date of registration followed by “retrospectively registered”Not applicable.AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology testing is key for assessing seroprevalence and antibody response post-vaccination in immunocompromised patients. Here we performed a comparison between two high-throughput nucleocapsid assays (Abbott SARS-CoV-2 IgG and Roche Elecsys Anti-SARS-CoV-2) and The Binding Site (TBS) anti-Spike IgG/A/M-SARS-CoV-2 ELISA kit. 236 samples were collected across 2 sites, Portsmouth Hospital University NHS Trust (PHU) and The Dudley Group NHS Trust. We derived concordance, agreement and assay performance as well as using receiver operating characteristic (ROC) curves to redefine the assay threshold of the Abbott assay. Result concordance between the Abbott and TBS was 66%. Discrepant samples were analysed using the Roche assay which showed 100% agreement with the TBS assay. In samples analysed >58 days post-PCR, the sensitivity of Abbott and Roche was 100%. In samples analysed >100 days post-PCR the sensitivity of the Abbott assay dropped to 77.2% but remained at 100% for the Roche assay. A redefined Abbott threshold of 0.64 increased the sensitivity to 90% giving results similar to the Roche and TBS assays. In conclusion, this study demonstrated Abbott assay had a lower sensitivity in comparison to TBS and Roche. This study established TBS can be implemented as a viable alternative for SARS-CoV-2 serology testing where high-throughput assays are not available on site. Furthermore, anti-spike assays, such as TBS, could be used to monitor vaccination responses to deduce SARS-CoV-2 population-immunity. Further optimisation studies are required to evaluate the performance characteristics of these assays which could facilitate widescale sero-epidemiological surveillance.

Whipple’s disease (WD) is a rare infection due to Tropheryma whipplei. Following in-vitro cultivation of T. whipplei, an indirect-immunofluorescence serological assay (IFA) was developed. We tested the hypothesis that this assay could be used to either identify WD patients, or rule out WD, in patients in whom the diagnosis is being considered, based on the antibody titers of their IgM and IgG antibody responses. In this small study fourteen WD patients and 22 healthy volunteers’ sera were obtained from across Australia. All specimens were coded and de-identified before testing. A patient with an IgG antibody titer of ≤1:16 may have WD [sensitivity 57% (8/14) and specificity close to 100% (22/22)]. High IgM antibody titers (≥1:256) were more common in WD patients [sensitivity 50% (7/14) and specificity 86% (19/22)] than in controls. The area under Receiver-Operator-Characteristic curve for IgG in the IFA assay was 0.84 (95% CI 0.69-1.00). At an IgG antibody titer of ≤1:16 the Youden’s index was 0.57. WD patients’ under-produce IgG antibody to T.whipplei but are more likely to over-produce IgM antibodies. This screening IFA serological assay may be clinically useful in detecting those with a possible diagnosis of WD. Patients with an IgG antibody titer of ≤1:16 and an IgM antibody titer of ≥1:256 may have WD and should proceed to a tissue biopsy and PCR for confirmation. Further validation of this assay, by increasing the sample size, by testing it in patients with non-WD disease and trialing in other countries should be undertaken.

SARS-CoV-2 unprecedentedly threatens the public health at worldwide level. There is an urgent need to develop an effective vaccine within a highly accelerated time. Here, we present the most comprehensive S-protein-based linear B-cell epitope candidate list by combining epitopes predicted by eight widely-used immune-informatics methods with the epitopes curated from literature published between Feb 6, 2020 and July 10, 2020. We find four top prioritized linear B-cell epitopes in the hotspot regions of S protein can specifically bind with serum antibodies from horse, mouse, and monkey inoculated with different SARS-CoV-2 vaccine candidates or a patient recovering from COVID-19. The four linear B-cell epitopes can induce neutralizing antibodies against both pseudo and live SARS-CoV-2 virus in immunized wild-type BALB/c mice. This study suggests that the four linear B-cell epitopes are potentially important candidates for serological assay or vaccine development.

Coronavirus Disease 2019 (COVID-19) is a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While detection of SARS-CoV-2 by polymerase chain reaction with reverse transcription (RT-PCR) is currently used to diagnose acute COVID-19 infection, serological assays are needed to study the humoral immune response to SARS-CoV-2. SARS-CoV-2 IgG/A/M antibodies against SARS-CoV-2 spike (S) protein and its receptor-binding domain (RBD) were characterized using an enzyme-linked immunosorbent assay (ELISA) and assessed for their ability to neutralize live SARS-CoV-2 virus in recovered subjects who were RT-PCR-positive (n=153), RT-PCR-negative (n=55), and control samples collected pre-COVID-19 (n=520). Anti-SARS-CoV-2 antibodies were detected in 90.9% of resolved subjects up to 180 days post-symptom onset. Anti-S protein and anti-RBD IgG titers correlated (r= 0.5157 and r = 0.6010, respectively) with viral neutralization. Of the RT-PCR-positive subjects, 22 (14.3%) did not have anti-SARS-CoV-2 antibodies; and of those, 17 had RT-PCR cycle threshold (Ct) values >27, raising the possibility that these indeterminate results are from individuals who were not infected, or had mild infection that failed to elicit an antibody response. This study highlights the importance of serological surveys to determine population-level immunity based on infection numbers as determined by RT-PCR.

More than 300 SARS-COV-2 serological tests have recently been developed using either the nucleocapsid phosphoprotein (N), the spike glycoprotein subunit (S1), and more recently the receptor binding domain (RBD). Most of the assays report very good clinical performance characteristics in well-controlled clinical settings. However, there is a growing belief that good performance characteristics that are obtained during clinical performance trials might not be sufficient to deliver good diagnostic results in population-wide screens that are usually characterized with low seroprevalence. In this paper, we developed a serological assay against N, S1 and RBD using a bead-based multiplex platform and a rules-based computational approach to assess the performance of single and multi-antigen readouts in well-defined clinical samples and in a population-wide serosurvey from blood donors. Even though assays based on single antigen readouts performed similarly well in the clinical samples, there was a striking difference between the antigens on the population-wide screen. Asymptomatic individuals with low antibody titers and sub-optimal assay specificity might contribute to the large discrepancies in population studies with low seroprevalence. A multi-antigen assay requiring partial agreement between RBD, N and S1 readouts exhibited enhanced specificity, less dependency on assay cut-off values and an overall more robust performance in both sample settings. Our data suggest that assays based on multiple antigen readouts combined with a rules-based computational consensus can provide a more robust platform for routine antibody screening.One Sentence SummaryClinical and Population-level performance of single and multiplex SARS-CoV-2 serological assays.

The capacity to accurately diagnosis COVID-19 is essential for effective public health measures to manage the ongoing global pandemic, yet no presently available diagnostic technologies or clinical protocols can achieve full positive predictive value (PPV) and negative predictive value (NPV) performance. Two factors prevent accurate diagnosis: the failure of sampling methods (e.g., 40% false negatives from PCR testing of nasopharyngeal swabs) and sampling-time-dependent failures reflecting individual humoral responses of patients (e.g., serological testing outside of the sero-positive stage). Here, we report development of a diagnostic protocol that achieves full PPV and NPV based on a cohort of 500 confirmed COVID-19 cases, and present several discoveries about the sero-conversion dynamics throughout the disease course of COVID-19. The fundamental enabling technology for our study and diagnostic protocol—termed SANE, for Symptom (dpo)-Antibody-Nucleic acid-Epidemiological history—is our development of a peptide-protein hybrid microarray (PPHM) for COVID-19. The peptides comprising PPHMcovid-19 were selected based on clinical sample data, and give our technology the unique capacity to monitor a patient’s humoral response throughout the disease course. Among other assay-development related and clinically relevant findings, our use of PPHMcovid-19 revealed that 5% of COVID-19 patients are from an “early sero-reversion” subpopulation, thus explaining many of the mis-diagnoses we found in our comparative testing using PCR, CLIA, and PPHMcovid-19. Accordingly, the full SANE protocol incorporates orthogonal technologies to account for these patient variations, and successfully overcomes both the sampling method and sampling time limitations that have previously prevented doctors from achieving unambiguous, accurate diagnosis of COVID-19.

As SARS-CoV-2 continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Convalescent serum is being used for treatment and for isolation of patient-derived antibodies. However, currently there is not a simple means to estimate serum bulk neutralizing capability. Here we present an efficient competitive serological assay that can simultaneously determine an individual’s seropositivity against the SARS-CoV-2 Spike protein and estimate the neutralizing capacity of anti-Spike antibodies to block interaction with the human angiotensin converting enzyme 2 (ACE2) required for viral entry. In this ELISA-based assay, we present natively-folded viral Spike protein receptor binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then supplemented with soluble ACE2-Fc to compete for RBD-binding serum antibodies, and antibody binding quantified. Comparison of signal from untreated serum and ACE2-Fc-treated serum reveals the presence of antibodies that compete with ACE2 for RBD binding, as evidenced by loss of signal with ACE2-Fc treatment. In our test cohort of nine convalescent SARS-CoV-2 patients, we found all patients had developed anti-RBD antibodies targeting the epitope responsible for ACE2 engagement. This assay provides a simple and high-throughput method to screen patient sera for potentially neutralizing anti-Spike antibodies to enable identification of candidate sera for therapeutic use.

BackgroundRT-PCR test for identification of viral nucleic acid is the current standard diagnostic method for the diagnosis of COVID-19 disease but technical reasons limit the utilization of this assay onlarge scalescreenings.MethodWe verified in a consecutive series of 191 symptomatic patients the clinical information that new rapid serological colorimetric test qualitatively analyzing IgM/IgG expression can provide with respect to standard assay and with respect to clinical outcome of patients.ResultsRapid serological test showed a sensitivity of 30% and a specificity of 89% with respect to the standard assay but, interestingly, these performances improve after 8 days of symptoms appearance. After 10 days of symptoms the predictive value of rapid serological test is higher than that of standardassay. When the behaviour of the two immunoglobulins was evaluated with respect to time length of symptoms appaerance, no significant difference in immunoglobulins behaviour was shown.ConclusionsThe rapid serological test analyzed in the present study is candidate to provide information on immunoreaction of the subject to COVID-19 exposure.

BackgroundThe outbreak of the recently emerged novel corona virus disease 2019 (COVID-19) poses a challenge for public health laboratories. We aimed to evaluate the diagnostic value of serological assay for SARS-CoV-2.MethodsA newly-developed ELISA assay for IgM and IgG antibodies against N protein of SARS-CoV-2 were used to screen the serums of 238 admitted hospital patients with confirmed or suspected SARS-CoV-2 infection from February 6 to February 14, 2020. SARS-CoV-2 RNA was detected by real time RT-PCR on pharyngeal swab specimens.FindingsOf the 238 patients, 194 (81.5%) were detected to be antibody (IgM and/or IgG) positive, which was significantly higher than the positive rate of viral RNA (64.3%). There was no difference in the positive rate of antibody between the confirmed patients (83.0%, 127/153) and the suspected patients (78.8%, 67/85) whose nucleic acid tests were negative. After the patients were defined to the different stages of disease based on the day when the test samples were collected, the analysis results showed that the antibody positive rates were very low in the first five days after initial onset of symptoms, and then rapidly increased as the disease progressed. After 10 days, the antibody positive rates jumped to above 80% from less than 50%. On the contrary, the positive rates of viral RNA kept above 60% in the first 11 days after initial onset of symptoms, and then rapidly decreased. In addition, half of the suspected patients with symptoms for 6-10 days were detected to be antibody positive.InterpretationThe suspected patients were most likely infected by SARS-CoV-2. Before the 11th day after initial onset of symptoms, nucleic acid test is important for confirmation of viral infection. The combination of serological assay can greatly improve the diagnostic efficacy. After that, the diagnosis for viral infection should be majorly dependent on serological assay.

IntroductionScrub typhus is a vector borne zoonotic disease caused by Orientia tsutsugamushi, endemic to tsutsugamushi triangle. As the characteristic eschar is not always present, laboratory testing especially serological assay are the main stay of diagnosis.Materials and methodsA total of 346 well-characterized sera from normals and patients with scrub typhus, malaria, dengue, enteric fever and gram negative septicaemia were tested for IgM antibodies by ST IgM ELISA and ST Ig M ICTResultsThe sensitivity and specificity of Scrub typhus IgM ICT and ELISA were 98.7, 96.3 and 97.4, 99.3 respectively. The IgM ICT and ELISA had a excellent concordance (99%) and a very high negative predictive value.ConclusionThe findings from this study suggest that IgM ICT and IgM ELISA can be used interchangeably for serodiagnosis of scrub typhus in resource poor settings.

BackgroundNo serological assay has 100% sensitivity. Statistically, the concentration of specific antibodies against antigens of parasites decreases with the duration of infection. This can result in false negative outputs of diagnostic tests for the subjects with old infectiong, e.g., for individuals infected in childhood. When a property of seronegative and seropositive subjects is compared under these circumstances, the statistical tests can detect no significant difference between these two groups of subjects, despite the fact that infected and noninfected subjects differ. When the effect of the infection has a cumulative character and subjects with an older infection (potential false negatives) are affected to a greater degree, we can even get paradoxical result of the comparison – the seronegative subjects have on average lower value of certain traits, e.g. IQ, despite the infection having a negative effect on the trait. A permutation test for the contaminated data, implemented, e.g., in the program Treept or available as a comprehensibly commented R function in the supplement of this paper, can be used to reveal and to eliminate the effect of false negatives.MethodsWe used a Monte Carlo simulation in the program R to show that the permutation test implemented in the programs Treept and PTPT is a conservative test.ResultsWe showed that the test could provide false negative but not false positive results if the studied population contains no subpopulation of false negative subjects. We also introduced R version of the test expanded by skewness analysis, which helps to estimate the proportion of false negative subjects based on the assumption of equal data skewness in groups of healthy and infected individuals.ConclusionsBased on the results of simulations and our experience with empirical studies we recommend the usage of permutation test for contaminated data whenever seronegative and seropositive individuals are compared.