用于测定母乳中SARS-CoV-2抗体活性的结合与中和试验的开发及一致性
Development and Concordance of Binding and Neutralizing Assays to Determine SARS-CoV-2 Antibody Activity in Human Milk.
作者信息
Shriver Mallory C, Milletich Patricia L, Moreno Alberto, Larsen Sasha E, Posavad Christine M, Berube Bryan J, Wali Bushra, Ellis Madison, Manning Kelly, Moore Kathryn M, Zhu Zhiyi, Grewal Nimrit, Cadena Ines A, Cardemil Cristina V, Munoz Flor M, Neuzil Kathleen M, Coler Rhea N, Suthar Mehul S, Pasetti Marcela F
机构信息
Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, Maryland.
Center for Childhood Infections and Vaccines, Children's Healthcare of Atlanta, Division of Infectious Diseases, Department of Pediatrics, Emory Vaccine Center, Emory University, Atlanta, Georgia.
出版信息
Pathog Immun. 2025 May 22;10(2):97-121. doi: 10.20411/pai.v10i2.799. eCollection 2025.
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.
背景
母体免疫通过母乳为婴儿提供疫苗特异性免疫力。多项研究报告了感染和/或接种疫苗的女性的人母乳（HBM）中存在严重急性呼吸综合征冠状病毒2（SARS-CoV-2）抗体。然而，关于所使用方法的分析性能、一致性和质量的信息有限。需要标准化和严格的检测方法来满足临床研究终点并进行跨研究比较。
方法
我们优化了高通量多重免疫测定法，用于定量HBM中的SARS-CoV-2免疫球蛋白（Ig）G和IgA，并测定了236名接种SARS-CoV-2疫苗（感染和未感染）和50名疫情前（未接触）哺乳期妇女的HBM样本中的抗体水平。此外，使用活病毒蚀斑减少中和试验和假病毒试验，对75名接种疫苗（感染和未感染）妇女的SARS-CoV-2 HBM子集进行了SARS-CoV-2中和活性检测。检查了SARS-CoV-2结合与活病毒中和结果之间的一致性。
结果
多重SARS-CoV-2检测具有足够的分析灵敏度、重复性、精密度和检测线性，可可靠地定量HBM中的IgG和IgA。确定了刺突蛋白和核衣壳特异性IgG和IgA的阳性阈值；IgG以高灵敏度和特异性区分SARS-CoV-2免疫HBM的阳性/阴性，而IgA反应性存在重叠。观察到活SARS-CoV-2与假病毒中和活性之间存在强相关性。HBM刺突蛋白IgA与中和滴度高度相关。
结论
使用标准化和严格的检测方法测定了HBM中的SARS-CoV-2结合和中和抗体活性。确定了SARS-CoV-2疫苗接种和感染的HBM阳性临界值。这里描述的方法和途径可应用于其他病原体和粘膜分泌物。
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