Development of a novel serological assay for the detection of mpox infection in vaccinated populations.

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 104 nonendemic locations worldwide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

Neutralizing Antibody Responses in COVID-19 Convalescent Sera.

Passive transfer of antibodies from COVID-19 convalescent patients is being used as an experimental treatment for eligible patients with SARS-CoV-2 infections. The United States Food and Drug Administration's (FDA) guidelines for convalescent plasma initially recommended target antibody titers of 160. We evaluated SARS-CoV-2 neutralizing antibodies in sera from recovered COVID-19 patients using plaque reduction neutralization tests (PRNT) at moderate (PRNT50) and high (PRNT90) stringency thresholds. We found that neutralizing activity significantly increased with time post symptom onset (PSO), reaching a peak at 31-35 days PSO. At this point, the number of sera having neutralizing titers of at least 160 was approximately 93% (PRNT50) and approximately 54% (PRNT90). Sera with high SARS-CoV-2 antibody levels (>960 enzyme-linked immunosorbent assay titers) showed maximal activity, but not all high-titer sera contained neutralizing antibody at FDA recommended levels, particularly at high stringency. These results underscore the value of serum characterization for neutralization activity.

Development of Zika Virus Serological Testing Strategies in New York State.

The recent outbreak of Zika virus (ZIKV) in the Americas has challenged diagnostic laboratory testing strategies. At the Wadsworth Center, ZIKV serological testing was performed for over 10,000 specimens, using a combination of an enzyme-linked immunosorbent assay (ELISA) for IgM antibodies (Abs) to ZIKV, a polyvalent microsphere immunoassay (MIA) to detect Abs broadly reactive with flaviviruses, and a plaque reduction neutralization test (PRNT) for further testing. Overall, 42% of patients showed serological evidence of flavivirus infection (primarily past dengue virus [DENV] infection), while 7% possessed IgM Abs to ZIKV and/or DENV. ZIKV IgM Abs typically arose within 3 to 4 days, with only one instance of duration beyond 100 days after reported symptoms. PRNT analysis of 826 IgM-positive specimens showed 7% positive neutralization to ZIKV alone, 9% to DENV alone, and 85% to both ZIKV and DENV. Thus, the extensive Ab cross-reactivity among flaviviruses significantly reduced the value of performing PRNT analysis, especially when a traditional paired serum algorithm with viral neutralization titering was used. Nevertheless, the finding of a negative ZIKV result by PRNT was invaluable for reassuring both physicians and patients. The MIA detected both IgM and IgG, which enabled us to identify patients who presented without IgM anti-ZIKV Abs but still had ZIKV-specific neutralizing Abs. On the basis of these results, a new algorithm, which included an IgM Ab capture (MAC)-ELISA to detect recent infection, a flavivirus MIA to identify patients no longer producing IgM, and a single-dilution PRNT for ZIKV exclusion and occasional discrimination of ZIKV and DENV, was implemented.

Competition-induced criticality in a model of meme popularity.

Heavy-tailed distributions of meme popularity occur naturally in a model of meme diffusion on social networks. Competition between multiple memes for the limited resource of user attention is identified as the mechanism that poises the system at criticality. The popularity growth of each meme is described by a critical branching process, and asymptotic analysis predicts power-law distributions of popularity with very heavy tails (exponent α<2, unlike preferential-attachment models), similar to those seen in empirical data.

Inflammatory and Autoimmune Aspects of Multisystem Inflammatory Syndrome in Children (MIS-C): A Prospective Cohort Study.

Multisystem Inflammatory Syndrome in Children (MIS-C) is a potentially life-threatening complication of COVID-19. The pathophysiological mechanisms leading to severe disease are poorly understood. This study leveraged clinical samples from a well-characterized cohort of children hospitalized with COVID-19 or MIS-C to compare immune-mediated biomarkers. Our objective was to identify selected immune molecules that could explain, in part, why certain SARS-CoV-2-infected children developed MIS-C. We hypothesized that type-2 helper T cell-mediated inflammation can elicit autoantibodies, which may account for some of the differences observed between the moderate-severe COVID-19 (COVID+) and MIS-C cohort. We enumerated blood leukocytes and measured levels of selected serum cytokines, chemokines, antibodies to COVID-19 antigens, and autoantibodies in children presenting to an academic medical center in Connecticut, United States. The neutrophil/lymphocyte and eosinophil/lymphocyte ratios were significantly higher in those in the MIS-C versus COVID+ cohort. IgM and IgA, but not IgG antibodies to SARS-CoV-2 receptor binding domain were significantly higher in the MIS-C cohort than the COVID+ cohort. The serum levels of certain type-2 cytokines (interleukin (IL)-4, IL-5, IL-6, IL-8, IL-10, IL-13, and IL-33) were significantly higher in children with MIS-C compared to the COVID+ and SARS-CoV-2-negative cohorts. IgG autoantibodies to brain antigens and pentraxin were higher in children with MIS-C compared to SARS-CoV-19-negative controls, and children with MIS-C had higher levels of IgG anti-contactin-associated protein-like 2 (caspr2) compared to the COVID+ and SARS-CoV-19-negative controls. We speculate that autoimmune responses in certain COVID-19 patients may induce pathophysiological changes that lead to MIS-C. The triggers of autoimmunity and factors accounting for type-2 inflammation require further investigation.

Development of a biomarker signature using grating-coupled fluorescence plasmonic microarray for diagnosis of MIS-C.

Multisystem inflammatory syndrome in children (MIS-C) is a rare but serious condition that can develop 4-6 weeks after a school age child becomes infected by SARS-CoV-2. To date, in the United States more than 8,862 cases of MIS-C have been identified and 72 deaths have occurred. This syndrome typically affects children between the ages of 5-13; 57% are Hispanic/Latino/Black/non-Hispanic, 61% of patients are males and 100% have either tested positive for SARS-CoV-2 or had direct contact with someone with COVID-19. Unfortunately, diagnosis of MIS-C is difficult, and delayed diagnosis can lead to cardiogenic shock, intensive care admission, and prolonged hospitalization. There is no validated biomarker for the rapid diagnosis of MIS-C. In this study, we used Grating-coupled Fluorescence Plasmonic (GCFP) microarray technology to develop biomarker signatures in pediatric salvia and serum samples from patients with MIS-C in the United States and Colombia. GCFP measures antibody-antigen interactions at individual regions of interest (ROIs) on a gold-coated diffraction grating sensor chip in a sandwich immunoassay to generate a fluorescent signal based on analyte presence within a sample. Using a microarray printer, we designed a first-generation biosensor chip with the capability of capturing 33 different analytes from 80  µ L of sample (saliva or serum). Here, we show potential biomarker signatures in both saliva and serum samples in six patient cohorts. In saliva samples, we noted occasional analyte outliers on the chip within individual samples and were able to compare those samples to 16S RNA microbiome data. These comparisons indicate differences in relative abundance of oral pathogens within those patients. Microsphere Immunoassay (MIA) of immunoglobulin isotypes was also performed on serum samples and revealed MIS-C patients had several COVID antigen-specific immunoglobulins that were significantly higher than other cohorts, thus identifying potential new targets for the second-generation biosensor chip. MIA also identified additional biomarkers for our second-generation chip, verified biomarker signatures generated on the first-generation chip, and aided in second-generation chip optimization. Interestingly, MIS-C samples from the United States had a more diverse and robust signature than the Colombian samples, which was also illustrated in the MIA cytokine data. These observations identify new MIS-C biomarkers and biomarker signatures for each of the cohorts. Ultimately, these tools may represent a potential diagnostic tool for use in the rapid identification of MIS-C.

Development of a Novel Serological Assay for the Detection of Mpox Infection in Vaccinated Populations.

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 103 non-endemic locations world-wide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay (MIA) using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important diagnostic tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

Mpox vaccine and infection-driven human immune signatures: an immunological analysis of an observational study.

BACKGROUND: Monkeypox virus has recently infected more than 88 000 people, raising concerns about our preparedness against this emerging viral pathogen. Licensed and approved for mpox, the JYNNEOS vaccine has fewer side-effects than previous smallpox vaccines and has shown immunogenicity against monkeypox in animal models. This study aims to elucidate human immune responses to JYNNEOS vaccination compared with mpox-induced immunity. METHODS: Peripheral blood mononuclear cells and sera were obtained from ten individuals vaccinated with one or two doses of JYNNEOS and six individuals diagnosed with monkeypox virus infection. Samples were obtained from seven individuals before vaccination to serve as a baseline. We examined the polyclonal serum (ELISA) and single B-cell (heavy chain gene and transcriptome data) antibody repertoires and T-cell responses (activation-induced marker and intracellular cytokine staining assays) induced by the JYNNEOS vaccine versus monkeypox virus infection. FINDINGS: All participants were men between the ages of 21 and 60 years, except for one woman in the group of mpox-convalescent individuals, and none had previous orthopoxvirus exposure. All mpox cases were mild. Vaccinee samples were collected 6-33 days after the first dose and 5-40 days after the second dose. Mpox-convalescent samples were collected 20-102 days after infection. In vaccine recipients, gene-level plasmablast and antibody responses were negligible and sera displayed moderate binding to recombinant orthopoxviral proteins (A29L, A35R, E8L, A30L, A27L, A33R, B18R, and L1R) and native proteins from the 2022 monkeypox outbreak strain. By contrast, recent monkeypox virus infection (within 20-102 days) induced robust serum antibody responses to monkeypox virus proteins and to native monkeypox virus proteins from a viral isolate obtained during the 2022 outbreak. JYNNEOS vaccine recipients presented robust orthopoxviral CD4+ and CD8+ T-cell responses. INTERPRETATION: Infection with monkeypox virus resulted in robust B-cell and T-cell responses, whereas immunisation with JYNNEOS elicited more robust T-cell responses. These data can help to inform vaccine design and policies for preventing mpox in humans. FUNDING: National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), and Icahn School of Medicine.

Anergy in CD4 memory T lymphocytes. II. Abrogation of TCR-induced formation of membrane signaling complexes.

Memory and naive CD4 T cells have unique regulatory pathways for self/non-self discrimination. A memory cell specific regulatory pathway was revealed using superantigens to trigger the TCR. Upon stimulation by bacterial superantigens, like staphylococcal enterotoxin B (SEB), TCR proximal signaling is impaired leading to clonal tolerance (anergy). In the present report, we show that memory cell anergy results from the sequestration of the protein tyrosine kinase ZAP-70 away from the TCR/CD3ζ chain. During SEB-induced signaling, ZAP-70 is excluded from both detergent-resistant membrane microdomains and the immunological synapse, thus blocking downstream signaling. We also show that the mechanism underlying memory cell anergy must involve Fyn kinase, given that the suppression of Fyn activity restores the movement of ZAP-70 to the immunological synapse, TCR proximal signaling, and cell proliferation. Thus, toleragens, including microbial toxins, may modulate memory responses by targeting the organizational structure of memory cell signaling complexes.

Superantigen-induced CD4 memory T cell anergy. I. Staphylococcal enterotoxin B induces Fyn-mediated negative signaling.

Memory CD4 T cells must provide robust protection for an organism while still maintaining self-tolerance. Superantigens reveal a memory cell-specific regulatory pathway, by which signaling through the TCR can lead to clonal tolerance (anergy). Here we show that the src kinase Fyn is a critical regulator of anergy in murine memory CD4 T cells induced by the bacterial superantigen staphylococcal enterotoxin B (SEB). Exposure to SEB results in impaired TCR signaling due to failed CD3/ZAP-70 complex formation. Further, signal transduction through the TCR remains similarly blocked when anergic memory cells are subsequently exposed to agonist peptide antigen. Pharmacological inhibition or genetic elimination of Fyn kinase reverses memory cell anergy, resulting in SEB-induced cell proliferation. The mechanism underlying impaired TCR signaling and subsequent memory cell anergy must involve a Fyn signaling pathway given that the suppression of Fyn activity restores CD3/ZAP-70 complex formation and TCR proximal signaling.

Evaluation of measles IgM antibody detection assays during the 2018-2019 outbreak in New York State.

Although measles was eliminated in the United States in 2000, a severe outbreak occurred between October 2018 and September 2019. New York was especially hard hit. Serology played an integral role in determining immune status (IgG) and identifying, along with molecular analyses, acute measles infections (IgM). Although an indirect immunofluorescence assay (IFA) was historically used by the New York State Department of Health for measles IgM detection, a higher throughput assay was needed to address the increased specimen numbers. Four commercial enzyme-linked immunosorbent assays (ELISAs) were evaluated for sensitivity and specificity in detecting measles IgM. Two ELISA formats were compared, indirect ELISA and IgM antibody capture. Both formats had comparable specificity as determined by cross-reactivity to non-measles specimens. Overall, the sensitivity of the capture ELISAs was greater than the indirect ELISAs and comparable to the indirect immunofluorescence assay with benefits regarding capacity, cost, and turnaround time.

Pharmacokinetics of convalescent plasma therapy in a COVID-19 patient with X-linked Agammaglobulinemia.

Convalescent plasma (CP) has been the first line of defense against numerous infectious diseases throughout history. The COVID-19 pandemic created a need for a quick, easily accessible, and effective treatment for severe disease and CP was able to meet that immediate need. The utility of CP warrants a better understanding of the pharmacokinetics of CP treatment. Here we present the case of a COVID-19 patient with a genetic deficiency in antibody production who received CP as a part of the treatment regimen. In depth serological analysis revealed a surprising lack of SARS-CoV-2 specific antibodies and reduced serum IgG following CP infusion. Our study highlights plasma dilution and accelerated antibody clearance as potential mechanisms for the variable efficacy of CP therapy.

COVID-19 Serology Control Panel Using the Dried-Tube Specimen Method.

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020. The immunoglobulin subtypes (total Ig, IgM, and IgG) and their respective reactivity to severe acute respiratory syndrome coronavirus 2 nucleocapsid, spike, and receptor-binding domain antigens of the samples were delineated and compared with the WHO International Standard to elucidate the exact binding antibody units of each CSCP sample and ensure the CSCP provides adequate reactivity for different types of serological test platforms. We distribute the CSCP as a kit with five coded tubes to laboratories around the world to be used to compare test kits for external quality assurance, for harmonizing laboratory testing, and for use as training materials for laboratory workers.

Harmonization of Multiple SARS-CoV-2 Reference Materials Using the WHO IS (NIBSC 20/136): Results and Implications.

Background: There is an urgent need for harmonization between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology platforms and assays prior to defining appropriate correlates of protection and as well inform the development of new rapid diagnostic tests that can be used for serosurveillance as new variants of concern (VOC) emerge. We compared multiple SARS-CoV-2 serology reference materials to the WHO International Standard (WHO IS) to determine their utility as secondary standards, using an international network of laboratories with high-throughput quantitative serology assays. This enabled the comparison of quantitative results between multiple serology platforms. Methods: Between April and December 2020, 13 well-characterized and validated SARS-CoV-2 serology reference materials were recruited from six different providers to qualify as secondary standards to the WHO IS. All the samples were tested in parallel with the National Institute for Biological Standards and Control (NIBSC) 20/136 and parallel-line assays were used to calculate the relevant potency and binding antibody units. Results: All the samples saw varying levels of concordance between diagnostic methods at specific antigen-antibody combinations. Seven of the 12 candidate materials had high concordance for the spike-immunoglobulin G (IgG) analyte [percent coefficient of variation (%CV) between 5 and 44%]. Conclusion: Despite some concordance between laboratories, qualification of secondary materials to the WHO IS using arbitrary international units or binding antibody units per milliliter (BAU/ml) does not provide any benefit to the reference materials overall, due to the lack of consistent agreeable international unit (IU) or BAU/ml conversions between laboratories. Secondary standards should be qualified to well-characterized reference materials, such as the WHO IS, using serology assays that are similar to the ones used for the original characterization of the WHO IS.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Performance evaluation of antibody-based point-of-care devices intended for the identification of immune responses to SARS-CoV-2.

The novel coronavirus outbreak caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) was first identified in December of 2019 in Wuhan, China. The local outbreak quickly rose to pandemic level that has spread to more than 188 countries with more than 19 million cases and 732,467 deaths worldwide. The current recommendation for testing is RT-PCR based tests of nasopharyngeal or alternatively nasal- and/or oropharyngeal swabs that detects infection with SARS-CoV-2 to diagnose acute infection. However, there is an urgent need for a quick and accurate antibody-based point-of-care test method to quickly identify evidence of SARS-CoV-2 infection among people who might be missed through active case finding and surveillance efforts. Serology tests measure the presence of antibodies in serum after infection. Here we compared the performance characteristics of 6 commercially available antibody-based point-of-care devices and their potential for identification of individuals infected at some time by SARS-CoV-2.

Multiplexed detection and quantification of human antibody response to COVID-19 infection using a plasmon enhanced biosensor platform.

The 2019 SARS CoV-2 (COVID-19) pandemic has illustrated the need for rapid and accurate diagnostic tests. In this work, a multiplexed grating-coupled fluorescent plasmonics (GC-FP) biosensor platform was used to rapidly and accurately measure antibodies against COVID-19 in human blood serum and dried blood spot samples. The GC-FP platform measures antibody-antigen binding interactions for multiple targets in a single sample, and has 100% selectivity and sensitivity (n = 23) when measuring serum IgG levels against three COVID-19 antigens (spike S1, spike S1S2, and the nucleocapsid protein). The GC-FP platform yielded a quantitative, linear response for serum samples diluted to as low as 1:1600 dilution. Test results were highly correlated with two commercial COVID-19 antibody tests, including an enzyme linked immunosorbent assay (ELISA) and a Luminex-based microsphere immunoassay. To demonstrate test efficacy with other sample matrices, dried blood spot samples (n = 63) were obtained and evaluated with GC-FP, yielding 100% selectivity and 86.7% sensitivity for diagnosing prior COVID-19 infection. The test was also evaluated for detection of multiple immunoglobulin isotypes, with successful detection of IgM, IgG and IgA antibody-antigen interactions. Last, a machine learning approach was developed to accurately score patient samples for prior COVID-19 infection, using antibody binding data for all three COVID-19 antigens used in the test.

Serological analysis reveals an imbalanced IgG subclass composition associated with COVID-19 disease severity.

Coronavirus disease 2019 (COVID-19) is associated with a wide spectrum of disease presentation, ranging from asymptomatic infection to acute respiratory distress syndrome (ARDS). Paradoxically, a direct relationship has been suggested between COVID-19 disease severity and the levels of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, including virus-neutralizing titers. A serological analysis of 536 convalescent healthcare workers reveals that SARS-CoV-2-specific and virus-neutralizing antibody levels are elevated in individuals that experience severe disease. The severity-associated increase in SARS-CoV-2-specific antibody is dominated by immunoglobulin G (IgG), with an IgG subclass ratio skewed toward elevated receptor binding domain (RBD)- and S1-specific IgG3. In addition, individuals that experience severe disease show elevated SARS-CoV-2-specific antibody binding to the inflammatory receptor FcÉ£RIIIa. Based on these correlational studies, we propose that spike-specific IgG subclass utilization may contribute to COVID-19 disease severity through potent Fc-mediated effector functions. These results may have significant implications for SARS-CoV-2 vaccine design and convalescent plasma therapy.

Testing-on-a-probe biosensors reveal association of early SARS-CoV-2 total antibodies and surrogate neutralizing antibodies with mortality in COVID-19 patients.

The association of mortality with the early humoral response to SARS-CoV-2 infection within the first few days after onset of symptoms (DAOS) has not been thoroughly investigated partly due to a lack of sufficiently sensitive antibody testing methods. Here we report two sensitive and automated testing-on-a-probe (TOP) biosensor assays for SARS-CoV-2 viral specific total antibodies (TAb) and surrogate neutralizing antibodies (SNAb), which are suitable for clinical use. The TOP assays employ an RBD-coated quartz probe using a Cy5-Streptavidin-polysacharide conjugate to improve sensitivity and minimize interference. Disposable cartridges containing pre-dispensed reagents require no liquid manipulation or fluidics during testing. The TOP-TAb assay exhibited higher sensitivity in the 0-7 DAOS window than a widely used FDA-EUA assay. The rapid and automated TOP-SNAb correlated well with two well-established SARS-CoV-2 virus neutralization tests. The clinical utility of the TOP assays was demonstrated by evaluating early antibody responses in 120 SARS-CoV-2 RT-PCR positive adult hospitalized patients. Higher TAb and SNAb positivity rates and more robust antibody responses at patient's initial hospital presentation were seen in inpatients who survived COVID-19 than those who died in the hospital. Survival analysis using the Cox Proportional Hazards Model showed that patients who had negative TAb and/or SNAb at initial hospital presentation were at a higher risk of in-hospital mortality. Furthermore, TAb and SNAb levels at presentation were inversely associated with SARS-CoV-2 viral load based on concurrent RT-PCR testing. Overall, the sensitive and automated TAb and SNAb assays allow the detection of early SARS-CoV-2 antibodies which associate with mortality.