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. 2025 May 19;8(3):52.
doi: 10.3390/mps8030052.

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

Luca Rovetini  1 , Gianina Florentina Belciug  1 , Luisa Massai  1 , Francesca Nonne  1 , Renzo Alfini  1 , Heena Ranchod  2 , Denasha L Reddy  2 , Mariagrazia Molfetta  1 , Davide Oldrini  1 , Makrina Totsika  1 , Miren Iturriza  1 , Ziyaad Dangor  2 , Carlo Giannelli  1 , Shabir A Madhi  2 , Francesca Micoli  1 , Martina Carducci  1 , Omar Rossi  1
Affiliations

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

Luca Rovetini et al. Methods Protoc. .

Abstract

Klebsiella pneumoniae 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 Klebsiella pneumoniae 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 Klebsiella pneumoniae.

Keywords: IgG quantification; Klebsiella pneumoniae; Luminex; immunogenicity; multiplex assay; vaccine.

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Conflict of interest statement

This work was performed with funds from the Gates Foundation and with internal funding from GlaxoSmithKline Biologicals SA. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Gates Foundation. GSK Vaccines Institute for Global Health Srl is an affiliate of GlaxoSmithKline Biologicals SA. L.R., G.F.B., L.M., F.N., R.A., M.M., D.O., M.T., S.R., M.I., C.G., F.M., M.C. and O.R. are employees of the GSK group of companies. S.A.M. received funding from Pfizer, AstraZeneca, Minervax, Merk, and funds from Gates Foundation all fully unrelated to the study described in this project.

Figures

Figure 1
Figure 1
Standard curve of MFI versus arbitrary units (AUs) obtained against each specific antigen pre-calibration (A) and in RLU/mL post-calibration (B). Geomean of individual points and standard deviation are shown for each datapoint together with the 5PL logistic curve fit; 1 RLU/mL was defined as the reciprocal sera dilution giving a signal of 1000 MFI (dotted line) in a standard assay.
Figure 2
Figure 2
Monoplex and multiplex standard curves: multivalent human standard serum was incubated with single (dotted line) or multiplex (continue line) bead preparations, and the MFIs were compared.
Figure 3
Figure 3
Assay precision. A total of 24 repeated measurements of RLU/mL from single independently handled samples by one operator in four different experiments. Single repeats are represented by circle symbols (repeats on different days are shown in orange for run 1, green for run 2, blue for run 3, and pink for run 4). Geometric means and geometric standard deviations from all repeats of each sample are represented by the grey line.
Figure 4
Figure 4
Assay linearity regression analysis. Blue dots represent log(RLU/mL) obtained for specific diluted sera. Red line represents the linear regression trendline, and 95% CI and 95% PI are also reported.
See this image and copyright information in PMC

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