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Research Article | Clinical Science and Epidemiology

Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides

Danka Pavliakova, Peter C. Giardina, Soraya Moghazeh, Shite Sebastian, Maya Koster, Viliam Pavliak, Andrew McKeen, Roger French, Kathrin U. Jansen, Michael Pride
Patricia P. Wilkins, Editor
Danka Pavliakova
aVaccine Research and Development and Early Clinical Development Biostatistics, Pfizer Inc., Pearl River, New York, USA
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Peter C. Giardina
aVaccine Research and Development and Early Clinical Development Biostatistics, Pfizer Inc., Pearl River, New York, USA
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Soraya Moghazeh
aVaccine Research and Development and Early Clinical Development Biostatistics, Pfizer Inc., Pearl River, New York, USA
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Shite Sebastian
bAffinivax, Cambridge, Massachusetts, USA
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Maya Koster
aVaccine Research and Development and Early Clinical Development Biostatistics, Pfizer Inc., Pearl River, New York, USA
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Viliam Pavliak
aVaccine Research and Development and Early Clinical Development Biostatistics, Pfizer Inc., Pearl River, New York, USA
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Andrew McKeen
cBiotechnology Clinical Development Statistics, Pfizer Inc., Pearl River, New York, USA
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Roger French
cBiotechnology Clinical Development Statistics, Pfizer Inc., Pearl River, New York, USA
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Kathrin U. Jansen
aVaccine Research and Development and Early Clinical Development Biostatistics, Pfizer Inc., Pearl River, New York, USA
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Michael Pride
aVaccine Research and Development and Early Clinical Development Biostatistics, Pfizer Inc., Pearl River, New York, USA
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Patricia P. Wilkins
Parasitology Services
Roles: Editor
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DOI: 10.1128/mSphere.00128-18
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ABSTRACT

A Luminex-based direct immunoassay (dLIA) platform has been developed to replace the standardized pneumococcal enzyme-linked immunosorbent assay platform. The multiplex dLIA simultaneously measures the concentration of serum immunoglobulin G (IgG) antibodies specific for pneumococcal capsular polysaccharide (PnPS) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The assay uses poly-l-lysine (PLL)-conjugated PnPS, chemically coupled to spectrally distinct Luminex microspheres. Assay validation experiments were performed using residual human serum samples obtained from 13-valent pneumococcal conjugate vaccine (13vPnC) clinical studies. Assay results are expressed as IgG antibody concentrations in micrograms per milliliter using the international reference serum, 007sp. The lower limit of quantitation (LLOQ) for all serotypes covered in the 13-plex dLIA fell within the range of 0.002 to 0.038 µg/ml serum IgG. The difference between the lower limit and upper limit of the assay range was >500-fold for all serotypes, and assay variability was <20% relative standard deviation (RSD) for all serotypes. IgG antibody measurements were shown to be serotype-specific (some cross-reactivity was observed only between the structurally related serotypes 6A and 6B as well as 19A and 19F), and no interference was observed between the serotypes when the assay was performed in the 13-plex format compared to the singleplex assays. The 13-plex dLIA platform developed by Pfizer Inc. generates up to 143 test results in a single 96-well plate and is a suitable replacement of the enzyme-linked immunosorbent assay (ELISA) platform for evaluating vaccine clinical trials.

IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.

FOOTNOTES

    • Received March 12, 2018.
    • Accepted June 9, 2018.
  • For a companion article on this topic, see https://doi.org/10.1128/mSphere.00127-18.

  • Copyright © 2018 Pavliakova et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides
Danka Pavliakova, Peter C. Giardina, Soraya Moghazeh, Shite Sebastian, Maya Koster, Viliam Pavliak, Andrew McKeen, Roger French, Kathrin U. Jansen, Michael Pride
mSphere Aug 2018, 3 (4) e00128-18; DOI: 10.1128/mSphere.00128-18

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Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides
Danka Pavliakova, Peter C. Giardina, Soraya Moghazeh, Shite Sebastian, Maya Koster, Viliam Pavliak, Andrew McKeen, Roger French, Kathrin U. Jansen, Michael Pride
mSphere Aug 2018, 3 (4) e00128-18; DOI: 10.1128/mSphere.00128-18
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KEYWORDS

Luminex
development
multiplex
pneumococcal
validation

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