Development and Characterization of a Ten-Plex Assay to Measure <i>Klebsiella pneumoniae</i> Antigen-Specific IgG in Human Sera

Development and Characterization of a Ten-Plex Assay to Measure <i>Klebsiella pneumoniae</i> Antigen-Specific IgG in Human Sera

<i>Klebsiella pneumoniae</i> is a leading cause of nosocomial infections, neonatal sepsis, and childhood mortality worldwide. A drastic rise in antibiotic-resistant isolates poses an urgent threat to humanity, and the World Health Organization (WHO) has classified this as a critical-prio...

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Main Authors: Luca Rovetini, Gianina Florentina Belciug, Luisa Massai, Francesca Nonne, Renzo Alfini, Heena Ranchod, Denasha L. Reddy, Mariagrazia Molfetta, Davide Oldrini, Makrina Totsika, Miren Iturriza, Ziyaad Dangor, Carlo Giannelli, Shabir A. Madhi, Francesca Micoli, Martina Carducci, Omar Rossi
Format: Article
Language:English
Published: MDPI AG 2025-05-01
Series:Methods and Protocols
Subjects:
<i>Klebsiella pneumoniae</i>
vaccine
immunogenicity
IgG quantification
multiplex assay
Luminex
Online Access:https://www.mdpi.com/2409-9279/8/3/52
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Summary:<i>Klebsiella pneumoniae</i> is a leading cause of nosocomial infections, neonatal sepsis, and childhood mortality worldwide. A drastic rise in antibiotic-resistant isolates poses an urgent threat to humanity, and the World Health Organization (WHO) has classified this as a critical-priority antimicrobial-resistant (AMR) pathogen. Recent advancements in developing vaccines against <i>Klebsiella pneumoniae</i> have highlighted the lack of standardized assays to evaluate immunogenicity, complicating comparison among different vaccines under development and the establishment of a serological threshold of risk reduction (SToRR). Here, we describe the development of a ten-plex multiplex assay to measure IgG against capsular polysaccharides (K2, K25, K102, K149), O antigens (O1v1, O1v2, O2v1, O2v2 and O5), and a conserved protein (MrkA). A standard curve was established by pooling human sera from naturally exposed subjects and then calibrated in terms of Relative Luminex Units/mL. The assay was fully characterized in terms of specificity, precision, linearity, and repeatability. This immunoassay demonstrates performance suitable for future clinical trials, as well as to perform sero-epidemiological studies to gain insights into naturally occurring immunity, potentially contributing to the establishment of a serological threshold of risk reduction against <i>Klebsiella pneumoniae</i>.
ISSN:2409-9279

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