Development and Concordance of Binding and Neutralizing Assays to Determine SARS-CoV-2 Antibody Activity in Human Milk

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Published: May 22, 2025
Keywords:
SARS-CoV-2 Antibodies, Breast Milk, Multiplex Assays, SARS-CoV-2 Neutralization

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Mallory C. Shriver
Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, Maryland
https://orcid.org/0009-0006-6536-3218
Patricia L. Milletich
Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, Maryland
https://orcid.org/0000-0002-7660-3057
Alberto Moreno
Center for Childhood Infections and Vaccines, Children's Healthcare of Atlanta, Division of Infectious Diseases, Department of Pediatrics, Emory Vaccine Center, Emory University; Emory National Primate Research Center; Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia
https://orcid.org/0000-0002-3031-7194
Sasha E. Larsen
Seattle Children's Research Institute, Center for Global Infectious Disease Research, Seattle, Washington
https://orcid.org/0000-0001-5034-9727
Christine M. Posavad
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington
https://orcid.org/0000-0003-3894-2784
Bryan J. Berube
Seattle Children's Research Institute, Center for Global Infectious Disease Research, Seattle, Washington
https://orcid.org/0000-0002-3806-040X
Bushra Wali
Center for Childhood Infections and Vaccines, Children's Healthcare of Atlanta, Division of Infectious Diseases, Department of Pediatrics, Emory Vaccine Center, Emory University; Emory National Primate Research Center, Atlanta, Georgia
https://orcid.org/0000-0001-9278-8744
Madison Ellis
Center for Childhood Infections and Vaccines, Children's Healthcare of Atlanta, Division of Infectious Diseases, Department of Pediatrics, Emory Vaccine Center, Emory University; Emory National Primate Research Center, Atlanta, Georgia
https://orcid.org/0000-0002-0325-5477
Kelly Manning
Center for Childhood Infections and Vaccines, Children's Healthcare of Atlanta, Division of Infectious Diseases, Department of Pediatrics, Emory Vaccine Center, Emory University; Emory National Primate Research Center, Atlanta, Georgia
https://orcid.org/0000-0002-6262-1190
Kathryn M. Moore
Center for Childhood Infections and Vaccines, Children's Healthcare of Atlanta, Division of Infectious Diseases, Department of Pediatrics, Emory Vaccine Center, Emory University; Emory National Primate Research Center, Atlanta, Georgia
https://orcid.org/0000-0002-8483-1148
Zhiyi Zhu
Seattle Children's Research Institute, Center for Global Infectious Disease Research, Seattle, Washington
Nimrit Grewal
Seattle Children’s Research Institute, Center for Global Infectious Disease Research, Seattle, Washington
Ines A. Cadena
Seattle Children's Research Institute, Center for Global Infectious Disease Research, Seattle, Washington
https://orcid.org/0009-0009-7345-7005
Cristina V. Cardemil
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland
https://orcid.org/0000-0002-5898-6154
Flor M. Munoz
Departments of Pediatrics and Molecular Virology & Microbiology, Baylor College of Medicine, and Texas Children’s Hospital, Houston, Texas
Kathleen M. Neuzil
Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, Maryland
https://orcid.org/0000-0001-9480-2714
Rhea N. Coler
Seattle Children's Research Institute, Center for Global Infectious Disease Research; University of Washington School of Medicine, Department of Pediatrics; University of Washington Department of Global Health, Seattle, Washington
https://orcid.org/0000-0001-6974-5666
Mehul S. Suthar
Center for Childhood Infections and Vaccines, Children's Healthcare of Atlanta, Division of Infectious Diseases, Department of Pediatrics, Emory Vaccine Center, Emory University; Emory National Primate Research Center; Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia
https://orcid.org/0000-0002-2686-8380
Marcela F. Pasetti
Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, Maryland
https://orcid.org/0000-0003-0894-4009

Abstract

Background: Maternal immunization provides vaccine-specific immunity to the infant via breast milk. Multiple studies have reported the presence of SARS-CoV-2 antibodies in human breast milk (HBM) from infected and/or vaccinated women. However, there is limited information on the analytical performance, consistency, and quality of the methods used. Standardized and
rigorous assays are needed to meet clinical study endpoints and for comparisons across studies.


Methods: We optimized high-throughput multiplex immunoassays for quantification of SARS-CoV-2 immunoglobulin (Ig)G and IgA in HBM and determined antibody levels in HBM samples from 236 SARS-CoV-2 vaccinated (infected and non-infected) and 50 pre-pandemic (unexposed) lactating women. Additionally, SARS-CoV-2 neutralizing activity was examined in a subset of 75 SARS-CoV-2 HBM from vaccinated (infected and non-infected) women using live virus focus reduction neutralization and pseudovirus assays. Concordance between SARS-CoV-2 binding and live virus neutralization outcomes was examined. 


Results: The multiplex SARS-CoV-2 assays had adequate analytical sensitivity, repeatability, precision, and assay linearity and were reliable for quantification of IgG and IgA in HBM. Positivity thresholds for Spike- and Nucleocapsid-specific IgG and IgA were established; IgG discriminated positive/negative SARS-CoV-2-immune HBM with high sensitivity and specificity, while IgA reactivity overlapped. A strong correlation was observed between live SARS-CoV-2 and pseudovirus neutralization activity. HBM Spike IgA and neutralization titers were highly correlated.


Conclusions: SARS-CoV-2 binding and neutralizing antibody activity in HBM was determined using standardized and rigorous assays. HBM positivity cutoff values for SARS-CoV-2 vaccination and infection were established. The methods and approach described here could be applied to other pathogens and mucosal secretions. 

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Vol. 10 No. 2 (2025): Volume 10, Number 2
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