Development of High-Throughput Multiplex Serology to Detect Serum Antibodies against Coxiella burnetii

The causative agent of Q fever, the bacterium Coxiella burnetii (C. burnetii), has gained increasing interest due to outbreak events and reports about it being a potential risk factor for the development of lymphomas. In order to conduct large-scale studies for population monitoring and to investigate possible associations more closely, accurate and cost-effective high-throughput assays are highly desired. To address this need, nine C. burnetii proteins were expressed as recombinant antigens for multiplex serology. This technique enables the quantitative high-throughput detection of antibodies to multiple antigens simultaneously in a single reaction. Based on a reference group of 76 seropositive and 91 seronegative sera, three antigens were able to detect C. burnetii infections. Com1, GroEL, and DnaK achieved specificities of 93%, 69%, and 77% and sensitivities of 64%, 72%, and 47%, respectively. Double positivity to Com1 and GroEL led to a combined specificity of 90% and a sensitivity of 71%. In a subgroup of seropositives with an increased risk for chronic Q fever, the double positivity to these markers reached a specificity of 90% and a sensitivity of 86%. Multiplex serology enables the detection of antibodies against C. burnetii and appears well-suited to investigate associations between C. burnetii infections and the clinical manifestations in large-scale studies.

SARS-CoV-2 seroprevalence and characteristics of post-infection immunity in a general population cohort study in Catalonia, Spain.

Sparse data exist on the complex natural immunity to SARS-CoV-2 at the population level. We applied a well-validated multiplex serology test in 5000 participants of a general population study in Catalonia in blood samples collected from end June to mid November 2020. Based on responses to fifteen isotype-antigen combinations, we detected a seroprevalence of 18.1% in adults (n=4740), and modeled extrapolation to the general population of Catalonia indicated a 15.3% seroprevalence. Antibodies persistedup to 9 months after infection. Immune profiling of infected individuals revealed that with increasing severity of infection (asymptomatic, 1-3 symptoms, ≥4 symptoms, admitted to hospital/ICU), seroresponses were more robust and rich with a shift towardsIgG over IgA and anti-spike over anti-nucleocapsid responses. Among seropositive participants, lower antibody levels were observed for those ≥60 years vs <60 years old and smokers vs non-smokers. Overweight/obese participants vs normal weight had higher antibody levels. Adolescents (13-15 years old) (n=260) showed aseroprevalence of 11.5%, were less likely to be tested seropositive compared to their parentsand had dominant anti-spike rather than anti-nucleocapside IgG responses. Our study provides an unbiased estimate of SARS-CoV-2 seroprevalence in Catalonia and new evidence on the durability and heterogeneity of post-infection immunity.

SARS-CoV-2 seroprevalence and characteristics of post-infection immunity in a general population cohort study in Catalonia, Spain.

Sparse data exist on the complex natural immunity to SARS-CoV-2 at the population level. We applied a well-validated multiplex serology test in 5000 participants of a general population study in Catalonia in blood samples collected from end June to mid November 2020. We detected a seroprevalence of 18.1% in adults (n=4740), and modeled extrapolation to the general population of Catalonia indicated a 15.3% seroprevalence. Antibodies persisted up to 9 months after infection. Immune profiling of infected individuals revealed that with increasing severity of infection (asymptomatic, 1-3 symptoms, ≥4 symptoms, admitted to hospital/ICU), seroresponses were more robust with a shift towards IgG over IgA and anti-spike over anti-nucleocapsid responses. Seropositive smokers showed lower seroresponses than non-smokers. In adolescents (n=260) seroprevalence was 11.5% and IgG anti-spike responses were dominant. Our study provides an unbiased estimate of SARS-CoV-2 seroprevalence in Catalonia and new evidence on the durability and heterogeneity of post-infection immunity.

multiSero: open multiplex-ELISA platform for analyzing antibody responses to SARS-CoV-2 infection

Serology has provided valuable diagnostic and epidemiological data on antibody responses to SARS-CoV-2 in diverse patient cohorts. Deployment of high content, multiplex serology platforms across the world, including in low and medium income countries, can accelerate longitudinal epidemiological surveys. Here we report multiSero, an open platform to enable multiplex serology with up to 48 antigens in a 96-well format. The platform consists of three components: ELISA-array of printed proteins, a commercial or home-built plate reader, and modular python software for automated analysis (pysero). We validate the platform by comparing antibody titers against the SARS-CoV-2 Spike, receptor binding domain (RBD), and nucleocapsid (N) in 114 sera from COVID-19 positive individuals and 87 pre-pandemic COVID-19 negative sera. We report data with both a commercial plate reader and an inexpensive, open plate reader (nautilus). Receiver operating characteristic (ROC) analysis of classification with single antigens shows that Spike and RBD classify positive and negative sera with the highest sensitivity at a given specificity. The platform distinguished positive sera from negative sera when the reactivity of the sera was equivalent to the binding of 1 ng mL-1 RBD-specific monoclonal antibody. We developed normalization and classification methods to pool antibody responses from multiple antigens and multiple experiments. Our results demonstrate a performant and accessible pipeline for multiplexed ELISA ready for multiple applications, including serosurveillance, identification of viral proteins that elicit antibody responses, differential diagnosis of circulating pathogens, and immune responses to vaccines.

From Multiplex Serology to Serolomics—A Novel Approach to the Antibody Response against the SARS-CoV-2 Proteome

The emerging SARS-CoV-2 pandemic entails an urgent need for specific and sensitive high-throughput serological assays to assess SARS-CoV-2 epidemiology. We, therefore, aimed at developing a fluorescent-bead based SARS-CoV-2 multiplex serology assay for detection of antibody responses to the SARS-CoV-2 proteome. Proteins of the SARS-CoV-2 proteome and protein N of SARS-CoV-1 and common cold Coronaviruses (ccCoVs) were recombinantly expressed in E. coli or HEK293 cells. Assay performance was assessed in a COVID-19 case cohort (n = 48 hospitalized patients from Heidelberg) as well as n = 85 age- and sex-matched pre-pandemic controls from the ESTHER study. Assay validation included comparison with home-made immunofluorescence and commercial enzyme-linked immunosorbent (ELISA) assays. A sensitivity of 100% (95% CI: 86–100%) was achieved in COVID-19 patients 14 days post symptom onset with dual sero-positivity to SARS-CoV-2 N and the receptor-binding domain of the spike protein. The specificity obtained with this algorithm was 100% (95% CI: 96–100%). Antibody responses to ccCoVs N were abundantly high and did not correlate with those to SARS-CoV-2 N. Inclusion of additional SARS-CoV-2 proteins as well as separate assessment of immunoglobulin (Ig) classes M, A, and G allowed for explorative analyses regarding disease progression and course of antibody response. This newly developed SARS-CoV-2 multiplex serology assay achieved high sensitivity and specificity to determine SARS-CoV-2 sero-positivity. Its high throughput ability allows epidemiologic SARS-CoV-2 research in large population-based studies. Inclusion of additional pathogens into the panel as well as separate assessment of Ig isotypes will furthermore allow addressing research questions beyond SARS-CoV-2 sero-prevalence.

A multiplex chemiluminescent immunoassay for serological profiling of COVID-19-positive symptomatic and asymptomatic patients

The COVID-19 pandemic affects more than 81 million people worldwide with over 1.7 million deaths. As the population returns to work, it is critical to develop tests that reliably detect SARS-CoV-2-specific antibodies. Here we present results from a multiplex serology test for assessing the antibody responses to COVID-19. In an initial large cohort, this test shows greater than 99% agreement with COVID-19 PCR test. In a second outpatient cohort consisting of adults and children in Colorado, the IgG responses are more robust in positive/symptomatic participants than in positive/asymptomatic participants, the IgM responses in symptomatic participants are transient and largely fall below the detection limit 30 days after symptom onset, and the levels of IgA against SARS-CoV-2 receptor binding domain are significantly increased in participants with moderate-to-severe symptoms compared to those with mild-to-moderate symptoms or asymptomatic individuals. Our results thus provide insight into serology profiling and the immune response to COVID-19.

Serological and hematological characteristics of Sjogren’s syndrome and dry eye syndrome patients using a novel immune serology technique

Objectives To compare hematologic and serological parameters among patients with Sjogren’s syndrome (SS), dry eye syndrome (DES) and controls, and validate a novel multiplex-serology method for identifying auto-antibodies in these populations. Methods In a clinic-based case-control study a total of 422 participants were recruited, including 91 with SS, 120 DES, and 211 controls (age and sex frequency-matched). We measured blood counts, anti-nuclear-antibodies (ANA), anti-SSA/SSB, anti-ribonucleoprotein (RNP), anti-double-stranded-DNA (DS-DNA), and rheumatoid factor (RF) using the “Immunodot” qualitative-ELISA assay. Immunoglobulins, C3 and C4 were measured by immune-fluorescence. Autoantibodies were also quantified with a newly-developed method using glutathione-S-transferase fusion proteins of SSA/Ro 52 and 60kD and SSB/La (multiplex-serology), measuring median fluorescence intensity (MFI). Results Among DES patients, only 2% (95%CI: 0.36–6.3) had positive immune serology. SS patients had lower lymphocyte, hemoglobin and C3 levels but higher prevalence of RF, ANA, anti-SSA/B and higher IgG and MFI levels, compared to DES and controls (P<0.001). Presence of anti-SSA/Ro-52kD was associated with SS [odds ratio (OR) = 2.05, 95% confidence interval (CI): 1.46–2.88]. Anti-SSB/La was inversely associated with DES (OR = 0.81, 95%CI: 0.65–1.00) compared to controls. Positivity to RF (adjusted for age, gender and ethnicity OR = 5.03, 95%CI: 1.78–14.21), ANA (OR = 14.75, 95%CI: 4.09–53.17), or combination of anti-SSA/B (OR = 20.97, 95%CI: 4.60–95.54) were more likely in SS compared to DES. The novel multiplex-serology method correctly identified anti-SSA/B autoantibodies by ELISA among SS, DES patients and controls (sensitivity = 1.0, negative-predictive-value = 1.0). Conclusions Serologic parameters distinguish SS from DES patients and controls. A newly-developed multiplex-serology technique may be useful to detect autoantibodies in large epidemiologic studies.

From multiplex serology to serolomics – a novel approach to the antibody response against the SARS-CoV-2 proteome

ABSTRACTBackgroundThe emerging SARS-CoV-2 pandemic entails an urgent need for specific and sensitive high-throughput serological assays to assess SARS-CoV-2 epidemiology. We therefore aimed at developing a fluorescent-bead based SARS-CoV-2 multiplex serology assay for detection of antibody responses to the SARS-CoV-2 proteome.MethodsProteins of the SARS-CoV-2 proteome and protein N of SARS-CoV-1 and common cold Coronaviruses (ccCoVs) were recombinantly expressed in E. coli or HEK293 cells. Assay performance was assessed in a Covid-19 case cohort (n=48 hospitalized patients from Heidelberg) as well as n=85 age- and sex-matched pre-pandemic controls from the ESTHER study. Assay validation included comparison with home-made immunofluorescence and commercial Enzyme-linked immunosorbent (ELISA) assays.ResultsA sensitivity of 100% (95% CI: 86%-100%) was achieved in Covid-19 patients 14 days post symptom onset with dual sero-positivity to SARS-CoV-2 N and the receptor-binding domain of the spike protein. The specificity obtained with this algorithm was 100% (95% CI: 96%-100%). Antibody responses to ccCoVs N were abundantly high and did not correlate with those to SARS-CoV-2 N. Inclusion of additional SARS-CoV-2 proteins as well as separate assessment of immunoglobulin (Ig) classes M, A, and G allowed for explorative analyses regarding disease progression and course of antibody response.ConclusionThis newly developed SARS-CoV-2 multiplex serology assay achieved high sensitivity and specificity to determine SARS-CoV-2 sero-positivity. Its high throughput ability allows epidemiologic SARS-CoV-2 research in large population-based studies. Inclusion of additional pathogens into the panel as well as separate assessment of Ig isotypes will furthermore allow addressing research questions beyond SARS-CoV-2 sero-prevalence.

Serology profile and effects of influenza vaccination on COVID-19-positive symptomatic and asymptomatic patients

The COVID-19 pandemic has affected more than 6.6 million people worldwide. As the population returns to work, it is critical to develop tests that can reliably detect SARS-CoV-2-specific antibodies. Here, we present the results of a novel multiplex serology test to assess the immune response to COVID-19 in an outpatient cohort consisting of adults and children in Colorado. The IgG response was more robust in positive/symptomatic participants than in positive/asymptomatic participants. The IgM response in symptomatic participants was transient and largely fell below the detection limit 30 days after symptom onset. Influenza vaccination gave rise to milder symptomology, but did not protect against contagion. These results provide novel insight into serology profiling and the immune response to COVID-19.

A translational multiplex serology approach to profile the prevalence of anti-SARS-CoV-2 antibodies in home-sampled blood

The COVID-19 pandemic has posed a tremendous challenge for the global community. We established a translational approach combining home blood sampling by finger-pricking with multiplexed serology to assess the exposure to the SARS-CoV-2 virus in a general population. The developed procedure determines the immune response in multiplexed assays against several spike (S, here denoted SPK), receptor binding domain (RBD) and nucleocapsid (NCP) proteins in eluates from dried capillary blood. The seroprevalence was then determined in two study sets by mailing 1000 blood sampling kits to random households in urban Stockholm during early and late April 2020, respectively. After receiving 55% (1097/2000) of the cards back within three weeks, 80% (878/1097) were suitable for the analyses of IgG and IgM titers. The data revealed diverse pattern of immune response, thus seroprevalence was dependent on the antigen, immunoglobulin class, stringency to include different antigens, as well as the required analytical performance. Applying unsupervised dimensionality reduction to the combined IgG and IgM data, 4.4% (19/435; 95% CI: 2.4%-6.3%) and 6.3% (28/443; 95% CI: 4.1%-8.6%) of the samples clustered with convalescent controls. Using overlapping scores from at least two SPK antigens, prevalence rates reached 10.1% (44/435; 95% CI: 7.3%-12.9%) in study set 1 and 10.8% (48/443; 95% CI: 7.9%-13.7%). Measuring the immune response against several SARS-CoV-2 proteins in a multiplexed workflow can provide valuable insights about the serological diversity and improve the certainty of the classification. Combining such assays with home-sampling of blood presents a viable strategy for individual-level diagnostics and towards an unbiased assessment of the seroprevalence in a population and may serve to improve our understanding about the diversity of COVID-19 etiology.One Sentence SummaryA multiplexed serology assay was developed to determine antibodies against SARS-CoV-2 proteins in home-sampled dried blood spots collected by finger pricking.