SARS-CoV-2 reactive antibodies in unexposed individuals revealed by a high sensitivity, low noise serologic assay

Yuen, Rachel; Steiner, Dylan; Pihl, Riley; Chavez, Elizabeth; Olson, Alex; Baird, Leemon C.; Korkmaz, Filiz; Urick, Patricia; Sagar, Manish; Berrigan, Jacob; Gummuluru, Suryaram; Corley, Ronald B.; Quillen, Karen; Belkina, Anna C.; Mostoslavsky, Gustavo; Rifkin, Ian R.; Kataria, Yachana; Cappione, Amedeo; Lin, Nina; Bhadelia, Nahid; Snyder‐Cappione, Jennifer

doi:10.1101/2020.09.15.20192765

Cited by 4 publications

(3 citation statements)

References 57 publications

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“…We also measured total IgG with an IgG assay kit (ThermoFisher, Cat. BMS2091) and IgA levels were determined with an in-house ELISA protocol (‘BU ELISA’) previously described (15) with a recombinant human IgA1 used as the standard (Bio-Rad, Cat. HCA189) Total IgG and IgA measurements were performed for all acute and convalescent subjects as well as 11 randomly chosen non-SLE unexposed subjects.…”

Section: Methodsmentioning

confidence: 99%

Distinct Autoimmune Antibody Signatures Between Hospitalized Acute COVID-19 Patients, SARS-CoV-2 Convalescent Individuals, and Unexposed Pre-Pandemic Controls

Bhadelia

Belkina

Olson

et al. 2021

Preprint

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Increasing evidence suggests that autoimmunity may play a role in the pathophysiology of SARS-CoV-2 infection during both the acute and ‘long COVID’ phases of disease. However, an assessment of autoimmune antibodies in convalescent SARS-CoV-2 patients has not yet been reported.MethodologyWe compared the levels of 18 different IgG autoantibodies (AABs) between four groups: (1) unexposed pre-pandemic subjects from the general population (n = 29); (2) individuals hospitalized with acute moderate-severe COVID-19 (n = 20); (3) convalescent SARS-COV-2-infected subjects with asymptomatic to mild viral symptoms during the acute phase with samples obtained between 1.8 and 7.3 months after infection (n = 9); and (4) unexposed pre-pandemic subjects with systemic lupus erythematous (SLE) (n = 6). Total IgG and IgA levels were also measured from subjects in groups 1-3 to assess non-specific pan-B cell activation.ResultsAs expected, in multivariate analysis, AABs were detected at much higher odds in SLE subjects (5 of 6, 83%) compared to non-SLE pre-pandemic controls (11 of 29, 38%) [odds ratio (OR) 19.4,95% CI, 2.0 – 557.0, p = 0.03]. AAB detection (percentage of subjects with one or more autoantibodies) was higher in SARS-CoV-2 infected convalescent subjects (7 of 9, 78%) [OR 17.4, 95% CI, 2.0 – 287.4, p = 0.02] and subjects with acute COVID-19 (12 of 20, 60%) compared with non-SLE pre-pandemic controls, but was not statistically significant among the latter [OR 1.8,95% CI, 0.6 – 8.1, p = 0.23]. Within the convalescent subject group, AABs were detected in 5/5 with reported persistent symptoms and 2/4 without continued symptoms (p = 0.17). The multivariate computational algorithm Partial Least Squares Determinant Analysis (PLSDA) was used to determine if distinct AAB signatures distinguish subject groups 1-3. Of the 18 autoantibodies measured, anti-Beta 2-Glycoprotein, anti-Proteinase 3-ANCA, anti-Mi-2 and anti-PM/Scl-100 defined the convalescent group; anti-Proteinase 3-ANCA, anti-Mi-2, anti-Jo-1 and anti-RNP/SM defined acute COVID-19 subjects; and anti-Proteinase 3-ANCA, anti-Mi-2, anti-Jo-1, anti-Beta 2-Glycoprotein distinguished unexposed controls. The AABs defining SARS-COV-2 infected from pre-pandemic subjects are widely associated with myopathies, vasculitis, and antiphospholipid syndromes, conditions with some similarities to COVID-19. Compared to pre-pandemic non-SLE controls, subjects with acute COVID-19 had higher total IgG concentration (p-value=0.006) but convalescent subjects did not (p-value=0.08); no differences in total IgA levels were found between groups.ConclusionsOur findings support existing studies suggesting induction of immune responses to self-epitopes during acute, severe COVID-19 with evidence of general B cell hyperactivation. Also, the preponderance of AAB positivity among convalescent individuals up to seven months after infection indicates potential initiation or proliferation, and then persistence of self-reactive immunity without severe initial disease. These results underscore the importance of further investigation of autoimmunity during SARS-CoV-2 infection and its role in the onset and persistence of post-acute sequelae of COVID-19.

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Section: Methodsmentioning

confidence: 99%

Distinct Autoimmune Antibody Signatures Between Hospitalized Acute COVID-19 Patients, SARS-CoV-2 Convalescent Individuals, and Unexposed Pre-Pandemic Controls

Bhadelia

Belkina

Olson

et al. 2021

Preprint

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“…We also thank Dr. Andrew Lodge at ThermoFisher Scientific for his advice regarding initial reagent selection and protocol steps. The manuscript has been released as a pre-print at MedRxIV, (Yuen et al) (46).…”

Section: Fundingmentioning

confidence: 99%

Novel ELISA Protocol Links Pre-Existing SARS-CoV-2 Reactive Antibodies With Endemic Coronavirus Immunity and Age and Reveals Improved Serologic Identification of Acute COVID-19 via Multi-Parameter Detection

et al. 2021

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The COVID-19 pandemic has drastically impacted work, economy, and way of life. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into pre-existing immunity, virus transmission dynamics, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient reliable detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. Very low optical densities from samples added to buffer coated wells at as low as a 1:5 dilution are reported using this ‘BU ELISA’ method. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (N) reactive antibodies (IgG, IgM, and/or IgA) in 44 and 100 percent of pre-pandemic subjects, respectively, and the magnitude of these antibodies tracked with antibody levels of analogous viral proteins from endemic coronavirus (eCoV) strains. The disease status (HIV, SLE) of unexposed subjects was not linked with SARS-CoV-2 reactive antibody levels; however, quantities were significantly lower in subjects over 70 years of age compared with younger counterparts. Also, we measured SARS-CoV-2 RBD- and N- specific IgM, IgG, and IgA antibodies from 29 SARS-CoV-2 infected individuals at varying disease states, including 10 acute COVID-19 hospitalized subjects with negative serology results by the EUA approved Abbott IgG chemiluminescent microparticle immunoassay. Measurements of SARS-CoV-2 RBD- and N- specific IgM, IgG, IgA levels measured by the BU ELISA revealed higher signal from 9 of the 10 Abbott test negative COVID-19 subjects than all pre-pandemic samples for at least one antibody specificity/isotype, implicating improved serologic identification of SARS-CoV-2 infection via multi-parameter, high sensitive antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and immunity and has promising implications for improved detection of all analytes measurable by this platform.

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“…However, detection of pathogen-specific antibodies that develop within days of infection is also a durable biomarker of prior exposure. The antibody-based assay has also been useful in identifying those who have been exposed to the virus 5,6 .…”

Section: Introductionmentioning

confidence: 99%

Dimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-19

Khan

Mishra

et al. 2021

Preprint

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Diagnostics has played a significant role in effective management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays. Thus far, limited knowledge exists about the antigenic properties of the N protein. In this paper, we demonstrate the significant impact of dimerization of SARS-CoV-2 nucleocapsid protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics of COVID-19. The expressed purified protein from E.coli consists of two forms, dimeric and monomeric forms, which have been further characterized by biophysical and immunological means. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form of the protein. These findings have also been confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. It is evident that use of the dimeric form will increase the sensitivity of the current nucleocapsid dependent ELISA for rapid COVID-19 diagnostic. Further, the results indicate that monitoring and maintaining of the monomer-dimer composition is critical for accurate and robust diagnostics.

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SARS-CoV-2 reactive antibodies in unexposed individuals revealed by a high sensitivity, low noise serologic assay

Cited by 4 publications

References 57 publications

Distinct Autoimmune Antibody Signatures Between Hospitalized Acute COVID-19 Patients, SARS-CoV-2 Convalescent Individuals, and Unexposed Pre-Pandemic Controls

Distinct Autoimmune Antibody Signatures Between Hospitalized Acute COVID-19 Patients, SARS-CoV-2 Convalescent Individuals, and Unexposed Pre-Pandemic Controls

Novel ELISA Protocol Links Pre-Existing SARS-CoV-2 Reactive Antibodies With Endemic Coronavirus Immunity and Age and Reveals Improved Serologic Identification of Acute COVID-19 via Multi-Parameter Detection

Dimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-19

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