---
title: "Pre-Omicron SARS-CoV-2 Immunity in the HIVE Cohort Using Multiplex ECL and Functional Assays"
id: "plos-one-22-evaluation-of-pre-omicron-population-immunity-for-sars-cov-2-in-the-household"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-22-evaluation-of-pre-omicron-population-immunity-for-sars-cov-2-in-the-household"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0360506"
published_at: "2026-10-15T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Pre-Omicron SARS-CoV-2 Immunity in the HIVE Cohort Using Multiplex ECL and Functional Assays
## Provenance & Clinical Metadata
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- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0360506)
- **Published At:** 2026-10-15T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The Household Influenza Vaccine Evaluation (HIVE) cohort provided serum samples collected between July 1 and December 31, 2021, before widespread Omicron circulation. The study nested a serosurvey within a long-running longitudinal household cohort with active ARI surveillance and documented vaccination and infection histories. - Researchers measured variant-specific IgG using a multiplex electrochemiluminescence (**ECL**) panel, assessed antibody blocking via **ACE2** inhibition multiplex assays, and compared results to **pseudoviral neutralization (PN)** titers. - A total of 156 serum samples from 117 participants were analyzed; most samples were from adults, and a majority of participants received COVID-19 vaccination during the sampling window. - Within-panel ECL IgG correlations across Spike and RBD antigens were high (Spearman 0.954–0.996). Cross-platform correlations were also strong: ECL versus PN (0.811–0.827) and ACE2 versus PN (0.877–0.953). - ROC analysis showed robust discrimination of vaccination status for ECL IgG (AUC 0.84–0.88; sensitivity 0.88–0.91; specificity 0.87–0.88). ACE2 inhibition assays had higher specificity but variable sensitivity and AUC across variants (AUC range reported by authors: 0.53–0.96). - BA.2-related antigens showed lower ACE2 assay performance and lower correlation with other variants in ACE2 inhibition metrics. - Population-level IgG concentrations rose with age and appeared to plateau in early adulthood, but estimates for young children were imprecise because of small sample sizes. - The study team noted strengths of the HIVE cohort (longitudinal surveillance and linked vaccination/infection data) and documented limits including small pediatric sample sizes and restricted individual-level data access.
## Clinical Analysis & Structured Key Points
Evaluation of pre-Omicron population immunity for SARS-CoV-2 in the Household Influenza Vaccine Evaluation (HIVE) cohort | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Peer Review Reader Comments Figures Figures Abstract Background Continued serosurvey for SARS-CoV-2 population immunity is critical for vaccine strain selection and identification of susceptible groups such as young children. We aim to evaluate the use of a high-throughput, low-volume assay to describe strain-specific IgG measurement in a longitudinally followed community based cohort. Methods The longitudinal Household Influenza Vaccine Evaluation study has observed around 300 households annually for acute respiratory illness. SARS-CoV-2 variant-specific IgG (including original, Delta, Beta, and Omicron variants) concentrations were measured using multiplex electrochemiluminescence (ECL), pseudoviral neutralization (PN), and ACE2 inhibition assays in participants who had serum samples drawn between July 1 and December 31, 2021. Spearman’s rank correlation coefficient assessed correlation within and between ECL, ACE2, and PN assays. Receiver operating characteristic (ROC) analysis evaluated the performance of ECL and ACE2 assays. Generalized additive mixed models estimated population immunity by age against SARS-CoV-2 variants. Results A total of 156 serum samples from 117 participants were selected for this study. Most (65.8%) were from adults and a majority (77.8%) received vaccination during the analysis period. High correlations were observed within ECL IgG assays (0.95–1.00), within ACE2 inhibition assays (0.70–0.99), and between assay platforms, including ECL versus PN (0.81–0.83) and ACE2 versus PN (0.88–0.95). ROC analysis demonstrated strong discrimination of vaccination status by ECL IgG assays (AUC: 0.84–0.88; sensitivity: 0.88–0.91; specificity: 0.87–0.88). ACE2 inhibition assays showed more variable performance (AUC: 0.53–0.96; sensitivity: 0.17–0.93; specificity: 0.91–1.00), with lower performance observed for some BA.2-related antigens. SARS-CoV-2 IgG concentrations generally increased with age and appeared to plateau in early adulthood, although estimates were imprecise in younger age groups because of small sample sizes. Conclusion We found a high correlation between ECL, ACE2, and PN across variants. Age-specific IgG quantities reflected behavior and infection histories collected by active surveillance. Citation: Yang Y, Juntila-Raymond C, Callear A, Smith M, Atkinson RK, Zhao Y, et al. (2026) Evaluation of pre-Omicron population immunity for SARS-CoV-2 in the Household Influenza Vaccine Evaluation (HIVE) cohort. PLoS One 21(10): e0360506. https://doi.org/10.1371/journal.pone.0360506 Editor: Esaki M. Shankar, Central University of Tamil Nadu, INDIA Received: June 26, 2026; Accepted: September 28, 2026; Published: October 15, 2026 Copyright: © 2026 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The individual-level data underlying this study cannot be made publicly available because they contain potentially identifying information from participants in an ongoing longitudinal household cohort and are subject to restrictions imposed by the University of Michigan Institutional Review Board. De-identified data necessary to reproduce the findings are available to qualified researchers upon reasonable request and approval by the University of Michigan. Data access requests may be directed to the corresponding authors. All author-generated code used for data processing, statistical analyses, and generation of the results and figures reported in this study is publicly available in the study’s GitHub repository: https://github.com/Jadeyyyp/HIVE-SC2-assay-manuscript-code . Funding: This project has been funded in whole with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N93021C00015, awarded to Emily T. Martin and Scott E. Hensley. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: We have no direct conflicts of interest to report related to this research. Outside of this work, A.S.M. has received CDC funding and grants from pharmaceutical consultations with Roche, Ltd. E.T.M. has received CDC funding and grants from pharmaceutical consultations with Merck and Co. S.E.H. is a co-inventor on patents that describe the use of nucleoside-modified mRNA as a vaccine platform. S.E.H reports receiving consulting fees from Sanofi, Pfizer, Lumen, Novavax, and Merck. S.E.H. and E.T.M. receive research funding from NIH UH2AI176136 awarded to Meso Scale Diagnostics, LLC, for unrelated work. None of these external activities or relationships were related to or provided support for the research reported in this manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Introduction COVID-19, caused by the SARS-CoV-2 virus, has shown a significant health burden globally, characterized by its rapid evolution and severe disease outcomes [ 1 ]. The immunoglobulin G (IgG) generated either post-vaccination or through natural infection is pivotal in preventing severe outcomes and, to some extent, subsequent infections [ 2 ]. Cross protection has been observed where IgG responses to one strain may cross protect against other strains [ 3 ]. Serological assays, which quantify specific immunoglobulins such as IgG neutralizing or functional antibodies, are important for understanding population immunity. Pseudovirus neutralization (PN) assays, a functional test that requires blocking viral entry and replication, typically focus on a single protein target per test and are technically challenging [ 4 ]. The complexity of immune responses to fast-evolving SARS-CoV-2 strains needs a broader approach. Multiplex electrochemiluminescence (ECL) assays assess antibody binding and can simultaneously detect multiple antigens. However, concerns remain that binding assays may not adequately assess the capacity to block infection and could potentially lack specificity against different variants. Another competition multiplex assay evaluates antibodies’ capacity to inhibit the binding of the angiotensin-converting enzyme 2 (ACE2) to SARS-CoV-2 antigen, thus analyzing antibody activity through ACE2 binding inhibition. Multiplex assays provide a robust and scalable solution for comprehensive immune profiling that can be used for large population-based serosuveys [ 5 , 6 ]. We conducted a multi-age community serosurvey of pre-Omicron antibody levels using a multiplex coronavirus immunoassay from Meso Scale Diagnostics (MSD, Rockville, USA) that utilizes multiplex electrochemiluminescence assay for the detection of antibodies against various protein targets, including the Spike protein, the Receptor Binding Domain (RBD) of the spike protein, and the nucleocapsid protein (N) of several SARS-CoV-2 strains. Additionally, our study used the ACE2 multiplex assay for antibodies that block the binding of ACE2 to Spike antigens from variants of SARS-CoV-2 to assess binding inhibition against various SARS-CoV-2 antigens. These assays were compared to PN tests, providing a comprehensive evaluation of the baseline antibody profile of participants in the longitudinal Household Influenza Vaccine Evaluation (HIVE) cohort conducted in Michigan before the spread of the Omicron variant. A major strength of the longitudinal HIVE cohort is the availability of repeated, high-quality immunologic measurements that capture longitudinal antibody immunity along with complete vaccination and infection history, making this a unique resource for nested serosurveys [ 7 – 12 ]. The period before widespread Omicron transmission (July 1 st – December 31 st 2021) offers a unique opportunity to study SARS-CoV-2 infection- and vaccine-induced immunity, as infection prevalence was low compared to the Omicron period, and vaccines had not yet been introduced to children. This created a clear distinction between infection-induced and vaccine-induced immunity, with few cases of infection in vaccinated individuals (i.e., “hybrid immunity”). Materials and methods Study population and serum samples Recruitment and retention procedures of HIVE study have been previously described [ 13 ]. Briefly, since 2010, eligible households must have ≥3 persons living at the same address with at least one child aged <10 years at the time of initial enrollment. In mid‐2020, eligibility was expanded to those with at least one child aged <21 years. Participants contributed blood specimens at enrollment, twice annually, pre- and post-flu and COVID-19 vaccination. For this analysis, serological samples were selected among HIVE participants who provided blood specimens between July 1 st and December 31 st, 2021. The HIVE study was approved by the University of Michigan Institutional Review Board. All participants were enrolled prospectively after completion of the informed consent process. Adults aged ≥18 years provided written informed consent for their own participation. For participants aged <18 years, written permission was obtained from a parent or legal guardian before participation. Children aged ≥7 years additionally provided verbal assent before study procedures were performed. The present analysis used serum specimens and study data collected under this approved longitudinal cohort protocol. Demographic information and household characteristics Each participant, or their parent or legal guardian, completed an enrollment survey detailing demographics, household characteristics, chronic medical conditions, COVID-19 vaccination history, and prior SARS-CoV-2 infection. Participants were surveyed annually to update demographic and household information. Weekly surveillance for COVID-19 symptoms was conducted. Households received weekly email reminders to report acute respiratory illnesses (ARIs) (see Supplementary Materials) at symptom onset (within 7 days post-onset) of qualifying ARIs by parents or through self-collection. All respiratory specimens were tested for SARS-CoV-2 using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR). Pre-Omicron Individual immunity profiles were classified into four categories: 1) Vaccination: participants who received at least the primary doses of COVID-19 vaccination as defined by the CDC; 2) Infection: participants with RT-PCR confirmed SARS-CoV-2 symptomatic infection before the emergence of Omicron; 3) Hybrid Immunity: participants who received at least the primary doses of COVID-19 vaccination and had RT-PCR confirmed SARS-CoV-2 infection before Omicron; 4) No vaccination, no infection. Serological assays Serum specimens were screened for IgG antibodies to the SARS-CoV-2 spike, RBD, and N protein, using a commercially available electrochemiluminescence (ECL) immunoassay kit Panel 29 (Spike antigens: original, B.1.351, B.1.617.2, BA.2, BA.2.12.1, BA.2.75, BA.4, BA.5)) and 30 (RBD antigens: original, B.1.1.7 B.1.351, BA.2, BA.2.12.1, BA.2.75, BA.4/5, delta. A result of ≥5000AU/mL N protein was considered seropositive [ 14 ]. Serum samples were diluted 1:5000 and 1:50,000 and run in duplicate on each plate and processed following the manufacturer's protocol [ 15 – 17 ]. Two serology controls (SC 1.2 and SC 1.3) provided by the manufacturer, containing known concentrations of IgG antibodies against the targets in the panel, were run in duplicate on each plate. Four independent runs for each sample were used to calculate coefficient of variation (CV) for quality control. Assay data were processed using standardized R code to resolve duplicates and reflex testing to higher dilutions (supplementary material). Competition ACE2 assays were used to evaluate the antibody inhibition capacity against SARS-CoV-2 antigens, specifically targeting spike and RBD proteins [ 18 , 19 ]. The percentage inhibition was calculated relative to the assay calibrator (maximum 100% inhibition) using the equation: PN assays were conducted to measure serum titers of neutralizing antibodies against Wild Type (WT) and BA.1 Spike strains, as previously described [ 20 , 21 ]. Briefly, VeroE6/TMPRSS2 cells were seeded at 2.5x10 4 cells/well in collagen-coated 96-well plates overnight. The following day, heat-inactivated sera were serially diluted 2-fold and mixed with ~300 focus-forming units per well of each VSVΔG-RFP SARS-CoV-2 pseudotype virus. A mouse anti-VSV G monoclonal antibody, 1E9F9, was spiked into virus-antibody mixtures at 600 ng/mL to ensure neutralization of any VSV-G carryover virus from pseudotype virus production. Virus-antibody mixtures were incubated for 1 h at 37 °C and then transferred to VeroE6/TMPRSS2 cells. After 22 hours postinfection at 37˚C, cells were washed with PBS, fixed with 4% PFA, and then blotted dry. Fluorescent foci were visualized and counted on an ImmunoSpot S6 analyzer. The focus reduction neutralization titer 50% (FRNT50) was measured as the greatest serum dilution at which the foci count was reduced by at least 50% relative to control cells that were infected with pseudotype virus in the absence of participant serum. FRNT50 titers for each sample were measured in two replicates. Statistical analysis Antibody concentrations were analyzed for individual Spike, RBD, and N antigens measured on the ECL panels, as well as PN titers against wild-type and BA.1 spike. Seropositivity for N was defined using the manufacturer-provided cutoff 5000AU/mL by ECL assay. Spearman correlation was used to compare antibody levels within and between ECL panel variants, and to compare between PN and ECL assays. Geometric mean concentration (GMC) and 95% confidence interval (95%CI) were used to assess the GMCs of antibody concentrations for each strain among age categories. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the ECL and ACE2 assay performance by antigen and protein targets. One serum sample per participant was included to ensure independence of observations. When participants contributed more than one serum sample during the study period, the earliest available sample was selected. Participants in the vaccinated-only and hybrid-immunity groups were classified as vaccinated, whereas participants in the prior-infection-only and unvaccinated-and-uninfected groups were classified as unvaccinated. ROC curves were generated, and the area under the curve (AUC) was calculated to assess discrimination. The optimal classification threshold was determined using Youden’s Index, which is the point on the receiver operating curve where sensitivity plus specificity is maximal, and aims to select an optimal threshold value for a diagnostic test [ 22 ]. At this threshold, sensitivity, specificity, and their 95% confidence intervals (CIs) were calculated using the Wilson score method. A generalized additive mixed model was used to fit antibody trends by age and SARS-CoV-2 strains pre-Omicron. All analyses were conducted using R version 4.0.38 [ 23 ], and plots were generated using the ggplot2 package. Results Participants and samples Among the participants enrolled in the HIVE cohort, 156 serum samples from 117 participants were selected for this study ( Table 1 ). Most participants were adults aged ≥18 years (n = 77, 65.8%), while 32 (27.4%) were aged 5–17 years and 7 (6.0%) were younger than 5 years. A slightly higher proportion of participants were female (n = 69, 59.5%) than male (n = 47, 40.5%). Participants were categorized as vaccinated only (n = 71, 60.7%), hybrid immunity (n = 20, 17.1%), unvaccinated and uninfected (n = 22, 18.8%), or prior infection only (n = 4, 3.4%). Among adults aged ≥18 years, 97.4% (75/77) had received COVID-19 vaccines, including 61 vaccinated-only participants and 14 with hybrid immunity. In contrast, among participants younger than 18 years, 53.8% (21/39) were unvaccinated and uninfected and 7.7% (3/39) had prior infection only. The majority of participants were White (n = 87, 75.0%), non-Hispanic (n = 112, 96.6%), and 34.5% (n = 40) reported at least one high-risk medical condition. Download: PNG larger image TIFF original image Table 1. Demographic characteristics of HIVE participants included in the pre-Omicron serosurvey, stratified by SARS-CoV-2 immune status. https://doi.org/10.1371/journal.pone.0360506.t001 Comparison of multiplex electrochemiluminescence and pseudo-neutralization Correlations between ECL measured IgG concentrations against Spike and RBD antigens were high across all strains, ranging from 0.954 to 0.996 ( S1 Fig ). Cross‑assay correlations were evaluated for matched variants only. Specifically, ECL Spike and RBD IgG concentrations were compared with PN titers for the same variant, and correlations were moderately high but lower than within‑panel ECL correlations for both Spike and RBD panels, ranging from 0.811 to 0.827. Spike and RBD ACE2 inhibition showed high correlation with PN titers for matched variants, ranging from 0.877 to 0.953. At comparable PN titer levels, ECL IgG concentrations were higher for the B.1.351 (Beta) and B.1.617.2 (Delta) variants compared to other variants. Antibody concentration relationship between antigens Among 156 serum samples, 17 (10.9%) were N-positive. Of these 17 N-positive samples, 8 were collected from participants with a documented PCR-confirmed SARS-CoV-2 infection before serum collection, whereas 9 were collected from participants without a documented prior PCR-confirmed infection. Spearman correlation coefficients were high across all antigen pairs: 0.991–0.999 (p < 0.05) for Spike IgG, 0.974–0.999 (p < 0.05) for RBD IgG, 0.866–0.992 (p < 0.05) for Spike ACE2, and 0.701–0.990 (p < 0.05) for RBD ACE2. Correlations between BA.2 and other variants were lower in ACE2 inhibition assays: 0.436–0.714 for Spike ACE2 and 0.329–0.435 for RBD ACE2 ( S1 Fig ). These results indicate small heterogeneity in antibody cross-reactivity across variants and assay types. Assay sensitivity and specificity ROC curve analysis was conducted to evaluate the diagnostic performance of each antigen for distinguishing vaccinated from unvaccinated participants. Among IgG assays, discrimination was consistently high, with AUC values ranging from 0.84–0.88 for IgG Spike and RBD antigens ( S1 Table ). Optimal thresholds determined using Youden's Index yielded high sensitivity (0.88–0.91) and specificity (0.87–0.88) across all IgG antigens. ACE2 inhibition assays generally demonstrated higher specificity (0.91–1.00) and AUC values ranging from 0.53 to 0.96, although performance varied substantially across variants. For Spike antigens, AUC values ranged from 0.64 for BA.2 to 0.96 for B.1.351, while RBD antigens ranged from 0.53 for BA.2.75 to 0.95 for Alpha and Delta variants. Sensitivity was more variable for ACE2 assays (0.17–0.93), with the lowest values observed for BA.2 Spike (0.42), BA.2 RBD (0.44), and BA.2.75 RBD (0.17). Pre-Omicron population antibody characteristics against SARS-CoV-2 SARS-CoV-2 ECL IgG concentrations generally increased with age and appeared to plateau around early adulthood across antigens ( Fig 1A ), although estimates at younger ages were imprecise because of the small number of participants, particularly among children aged <5 years. This pattern was consistent regardless of the specific antigen, indicating high cross-reactivity within the panel. Antibody concentrations in unvaccinated and uninfected participants remained consis
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