---
title: "Reconciling SARS-CoV-2 Neutralizing Antibody Titer Differences Across Assays"
id: "pubmed-42657887"
canonical_url: "https://medichelpline.com/clinical-feed/pubmed-42657887"
content_type: "clinical_feed_article"
specialty: "Critical Care"
source_name: "PubMed / NCBI"
source_url: "https://pubmed.ncbi.nlm.nih.gov/42657887/"
doi: "10.1128/jvi.00823-26"
published_at: "2026-09-22T00:00:00.000Z"
evidence_level: "Journal Article"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Reconciling SARS-CoV-2 Neutralizing Antibody Titer Differences Across Assays
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/pubmed-42657887
- **Specialty:** [Critical Care](https://medichelpline.com/clinical-feed/critical-care.md)
- **Primary Source:** PubMed / NCBI
- **Source URL:** [Original Journal Publication](https://pubmed.ncbi.nlm.nih.gov/42657887/)
- **DOI:** [10.1128/jvi.00823-26](https://doi.org/10.1128%2Fjvi.00823-26)
- **Published At:** 2026-09-22T00:00:00.000Z
- **Evidence Rating:** Journal Article
## Executive GIST (TL;DR)
- Neutralizing antibody titers against **SARS-CoV-2** are a correlate of protection and inform public health decisions, but no single standard neutralization assay exists, producing differing absolute titers between platforms. - The study compared 28 samples across three assay types: a live virus cytopathic effect (CPE)-based microneutralization assay, a live virus focus reduction (immunospot, **IS**) assay, and a pseudotyped virus luciferase reduction (**PV**) assay. - Strong correlations were observed between all assay pairings (Spearman ρ > 0.95; P < 0.0001 for all pairwise comparisons), indicating consistent relative ranking of samples across platforms. - Despite high correlation, absolute titers differed significantly between assays (paired t-tests P < 0.001). - The **IS** and **PV** assays, which measure the titer required to reduce foci or luminescence by 50%, produced the smallest difference: IS titers were 1.2-fold higher than PV (95% CI: 1.2–1.2). - The **CPE** assay produced substantially lower titers, measuring 5.5-fold lower than the IS assay (95% CI: 5.4–5.5). - The authors attribute key sources of these absolute differences to: (1) how the inoculum is considered — some assays measure neutralization on a **per virion** basis (half the infectious virions neutralized in IS and PV), whereas others measure **per well** neutralization (entire inoculum neutralized half the time in CPE); and (2) procedural differences such as removal of serum during the 5-day culture in the CPE assay, which limits neutralization to the inoculum rather than allowing ongoing antibody effects during viral replication. - These mechanistic explanations identify specific assay elements that affect measured neutralization and highlight the need for clearer, harmonized terminology and attention to assay design when comparing or standardizing neutralization results.
## Clinical Analysis & Structured Key Points
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Epub 2026 Aug 27. # Reconciliation of titer differences between SARS-CoV-2 neutralizing antibody assays [Eva Stadler](https://pubmed.ncbi.nlm.nih.gov/?term=Stadler+E&cauthor_id=42657887)[#](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-equal-contrib-explanation "Contributed equally")[ 1 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-affiliation-1 "Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, New South Wales, Australia."), [Francesca Mordant](https://pubmed.ncbi.nlm.nih.gov/?term=Mordant+F&cauthor_id=42657887)[#](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-equal-contrib-explanation "Contributed equally")[ 2 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-affiliation-2 "Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, Victoria, Australia."), [Matthew Gartner](https://pubmed.ncbi.nlm.nih.gov/?term=Gartner+M&cauthor_id=42657887)[ 2 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-affiliation-2 "Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, Victoria, Australia."), [Steffen S Docken](https://pubmed.ncbi.nlm.nih.gov/?term=Docken+SS&cauthor_id=42657887)[ 1 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-affiliation-1 "Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, New South Wales, Australia."), [Miles P Davenport](https://pubmed.ncbi.nlm.nih.gov/?term=Davenport+MP&cauthor_id=42657887)[#](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-equal-contrib-explanation "Contributed equally")[ 1 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-affiliation-1 "Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, New South Wales, Australia."), [David S Khoury](https://pubmed.ncbi.nlm.nih.gov/?term=Khoury+DS&cauthor_id=42657887)[#](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-equal-contrib-explanation "Contributed equally")[ 1 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-affiliation-1 "Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, New South Wales, Australia."), [Kanta Subbarao](https://pubmed.ncbi.nlm.nih.gov/?term=Subbarao+K&cauthor_id=42657887)[#](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-equal-contrib-explanation "Contributed equally")[ 2 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-affiliation-2 "Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, Victoria, Australia.")[ 3 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#full-view-affiliation-3 "WHO Collaborating Centre for Reference and Research on Influenza, VIDRL at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.") Affiliations Expand ### Affiliations * 1 Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, New South Wales, Australia. * 2 Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, Victoria, Australia. * 3 WHO Collaborating Centre for Reference and Research on Influenza, VIDRL at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia. # Contributed equally. * PMID: **42657887** * DOI: [ 10.1128/jvi.00823-26 ](https://doi.org/10.1128/jvi.00823-26) Item in Clipboard # Reconciliation of titer differences between SARS-CoV-2 neutralizing antibody assays Eva Stadler et al. J Virol. 2026. Show details Display options Display options Format Abstract PubMed PMID J Virol Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22J+Virol%22%5Bjour%5D&sort=date&sort_order=desc) * [ Search in NLM Catalog ](https://www.ncbi.nlm.nih.gov/nlmcatalog?term=%22J+Virol%22%5BTitle+Abbreviation%5D) * [ Add to Search ](https://pubmed.ncbi.nlm.nih.gov/42657887/) . 2026 Sep 22;100(9):e0082326. doi: 10.1128/jvi.00823-26. Epub 2026 Aug 27. ### Authors [Eva Stadler](https://pubmed.ncbi.nlm.nih.gov/?term=Stadler+E&cauthor_id=42657887)[#](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-equal-contrib-explanation "Contributed equally")[ 1 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-affiliation-1 "Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, New South Wales, Australia."), [Francesca Mordant](https://pubmed.ncbi.nlm.nih.gov/?term=Mordant+F&cauthor_id=42657887)[#](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-equal-contrib-explanation "Contributed equally")[ 2 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-affiliation-2 "Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, Victoria, Australia."), [Matthew Gartner](https://pubmed.ncbi.nlm.nih.gov/?term=Gartner+M&cauthor_id=42657887)[ 2 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-affiliation-2 "Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, Victoria, Australia."), [Steffen S Docken](https://pubmed.ncbi.nlm.nih.gov/?term=Docken+SS&cauthor_id=42657887)[ 1 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-affiliation-1 "Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, New South Wales, Australia."), [Miles P Davenport](https://pubmed.ncbi.nlm.nih.gov/?term=Davenport+MP&cauthor_id=42657887)[#](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-equal-contrib-explanation "Contributed equally")[ 1 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-affiliation-1 "Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, New South Wales, Australia."), [David S Khoury](https://pubmed.ncbi.nlm.nih.gov/?term=Khoury+DS&cauthor_id=42657887)[#](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-equal-contrib-explanation "Contributed equally")[ 1 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-affiliation-1 "Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, New South Wales, Australia."), [Kanta Subbarao](https://pubmed.ncbi.nlm.nih.gov/?term=Subbarao+K&cauthor_id=42657887)[#](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-equal-contrib-explanation "Contributed equally")[ 2 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-affiliation-2 "Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, Victoria, Australia.")[ 3 ](https://pubmed.ncbi.nlm.nih.gov/42657887/#short-view-affiliation-3 "WHO Collaborating Centre for Reference and Research on Influenza, VIDRL at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.") ### Affiliations * 1 Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, New South Wales, Australia. * 2 Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, Victoria, Australia. * 3 WHO Collaborating Centre for Reference and Research on Influenza, VIDRL at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia. # Contributed equally. * PMID: **42657887** * DOI: [ 10.1128/jvi.00823-26 ](https://doi.org/10.1128/jvi.00823-26) Item in Clipboard Full text links Cite Display options Display options Format Abstract PubMed PMID ## Abstract SARS-CoV-2 antibody neutralization is a correlate of protection for COVID-19 and has been used to guide public health decisions. However, there is no standard neutralization assay, and absolute titers differ between assays. To investigate mechanisms causing these absolute titer differences, we compared 28 samples with three assays: a live virus cytopathic effect (CPE)-based microneutralization assay, a live virus focus reduction (immunospot, IS) assay, and a pseudotyped virus luciferase expression reduction (pseudovirus, PV) assay. We found significant correlations between all assays (ρ > 0.95 and _P_ < 0.0001 for all pairwise comparisons), but also significant differences in absolute titers by assay (paired _t_ -tests _P_ < 0.001). IS and PV assays that assess neutralization as the titer required to reduce foci/luminescence by 50% had the smallest difference, with 1.2-fold higher IS assay titers (95% confidence interval, CI: 1.2-1.2). CPE assay titers were 5.5-fold lower (95% CI: 5.4-5.5) than in the IS assay. We show that these differences can be explained (i) by accounting for the size of inoculum being neutralized (half the infectious virions in the IS and PV assays, "per virion," and the entire inoculum half the time in the CPE assay, "per well") and (ii) by removing serum during the 5-day culture in the CPE assay (so serum can only neutralize the inoculum, rather than acting on the inoculum and slowing viral growth during culture). These findings highlight areas for more precise and harmonized terminology and particular assay elements that must be considered in efforts to harmonize neutralization assays. **Importance:** Protection against COVID-19 is related to how well serum antibodies neutralize SARS-CoV-2. This neutralization can be assessed using different assays, which often provide significantly different estimates. To better understand why assay results vary, we compared three commonly used types of neutralization assay. We found both strong correlations and significant differences in measured neutralization between the different assays. Differences in the type of virus (live SARS-CoV-2 virus compared to a pseudotyped virus with SARS-CoV-2 spike protein), how long cells, virus, and antibody were allowed to interact, and whether neutralization of a batch of virions ("per well" neutralization) or individual infectious virions ("per virion") was measured, all impacted virus neutralization. Understanding which factors have the largest impact on the differences between neutralization assays is an important first step toward harmonization of neutralization assays across the field. **Keywords:** SARS-CoV-2; assay differences; microneutralization; neutralization; plaque-reduction assay; pseudovirus. [PubMed Disclaimer](https://pubmed.ncbi.nlm.nih.gov/disclaimer/) ## Conflict of interest statement The authors declare no conflict of interest. ## MeSH terms * Animals Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22Animals%22%5BMeSH%5D&sort=date&sort_order=desc) * [ Search in MeSH ](https://www.ncbi.nlm.nih.gov/mesh?term=Animals) * [ Add to Search ](https://pubmed.ncbi.nlm.nih.gov/42657887/) * Antibodies, Neutralizing* / blood Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22Antibodies%2C+Neutralizing%2Fblood%22%5BMAJR%5D&sort=date&sort_order=desc) * [ Search in MeSH ](https://www.ncbi.nlm.nih.gov/mesh?term=Antibodies%2C+Neutralizing) * [ Add to Search ](https://pubmed.ncbi.nlm.nih.gov/42657887/) * Antibodies, Neutralizing* / immunology Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22Antibodies%2C+Neutralizing%2Fimmunology%22%5BMAJR%5D&sort=date&sort_order=desc) * [ Search in MeSH ](https://www.ncbi.nlm.nih.gov/mesh?term=Antibodies%2C+Neutralizing) * [ Add to Search ](https://pubmed.ncbi.nlm.nih.gov/42657887/) * Antibodies, Viral* / blood Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22Antibodies%2C+Viral%2Fblood%22%5BMAJR%5D&sort=date&sort_order=desc) * [ Search in MeSH ](https://www.ncbi.nlm.nih.gov/mesh?term=Antibodies%2C+Viral) * [ Add to Search ](https://pubmed.ncbi.nlm.nih.gov/42657887/) * Antibodies, Viral* / immunology Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22Antibodies%2C+Viral%2Fimmunology%22%5BMAJR%5D&sort=date&sort_order=desc) * [ Search in MeSH ](https://www.ncbi.nlm.nih.gov/mesh?term=Antibodies%2C+Viral) * [ Add to Search ](https://pubmed.ncbi.nlm.nih.gov/42657887/) * COVID-19* / diagnosis Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22COVID-19%2Fdiagnosis%22%5BMAJR%5D&sort=date&sort_order=desc) * [ Search in MeSH ](https://www.ncbi.nlm.nih.gov/mesh?term=COVID-19) * [ Add to Search ](https://pubmed.ncbi.nlm.nih.gov/42657887/) * COVID-19* / immunology Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22COVID-19%2Fimmunology%22%5BMAJR%5D&sort=date&sort_order=desc) * [ Search in MeSH ](https://www.ncbi.nlm.nih.gov/mesh?term=COVID-19) * [ Add to Search ](https://pubmed.ncbi.nlm.nih.gov/42657887/) * COVID-19* / virology Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22COVID-19%2Fvirology%22%5BMAJR%5D&sort=date&sort_order=desc) * [ Search in MeSH ](https://www.ncbi.nlm.nih.gov/mesh?term=COVID-19) * [ Add to Search ](https://pubmed.ncbi.nlm.nih.gov/42657887/) * Chlorocebus aethiops Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22Chlorocebus+aethiops%22%5BMeSH%5D&sort=date&sort_order=desc) * [ Search in MeSH ](https://www.ncbi.nlm.nih.gov/mesh?term=Chlorocebus+aethiops) * [ Add to Search ](https://pubmed.ncbi.nlm.nih.gov/42657887/) * Humans Actions * [ Search in PubMed ](https://pubmed.ncbi.nlm.nih.gov/?term=%22Humans%22%5BMeSH%5D&sort=date&sort_order=desc) * [ Search in MeSH ](https://www.ncbi.nlm.nih.gov/mesh?term=Humans)
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