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MICROBIAL IMMUNOLOGY

Serologic Assay To Quantify Human Immunoglobulin G Antibodies to the Staphylococcus aureus Iron Surface Determinant B Antigen

Michael D. Raedler, Samantha Heyne, Erica Wagner, Sheri K. Shalkowski, Susan Secore, Annaliesa S. Anderson, James Cook, Leslie Cope, Tessie McNeely, Mary Retzlaff, Jon Shanter, Leonard J. Rubinstein, Tina Green, N. Kartsonis, Mark T. Esser
Michael D. Raedler
1Wayne Clinical Support, Merck Research Laboratories, 466 Devon Park Dr., Wayne, Pennsylvania 19087-8630
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  • For correspondence: michael.raedler@ppdi.com
Samantha Heyne
1Wayne Clinical Support, Merck Research Laboratories, 466 Devon Park Dr., Wayne, Pennsylvania 19087-8630
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Erica Wagner
1Wayne Clinical Support, Merck Research Laboratories, 466 Devon Park Dr., Wayne, Pennsylvania 19087-8630
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Sheri K. Shalkowski
1Wayne Clinical Support, Merck Research Laboratories, 466 Devon Park Dr., Wayne, Pennsylvania 19087-8630
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Susan Secore
2Vaccine Basic Research, Merck Research Laboratories, West Point, Pennsylvania 19486
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Annaliesa S. Anderson
2Vaccine Basic Research, Merck Research Laboratories, West Point, Pennsylvania 19486
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James Cook
2Vaccine Basic Research, Merck Research Laboratories, West Point, Pennsylvania 19486
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Leslie Cope
2Vaccine Basic Research, Merck Research Laboratories, West Point, Pennsylvania 19486
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Tessie McNeely
2Vaccine Basic Research, Merck Research Laboratories, West Point, Pennsylvania 19486
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Mary Retzlaff
3Bioprocess Research and Development, Merck Research Laboratories, West Point, Pennsylvania 19486
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Jon Shanter
3Bioprocess Research and Development, Merck Research Laboratories, West Point, Pennsylvania 19486
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Leonard J. Rubinstein
5Infectious Disease/Vaccines, Merck Research Laboratories, Upper Gwynedd, Pennsylvania 19454
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Tina Green
4Non-Clinical Statistics, Merck Research Laboratories, West Point, Pennsylvania 19486
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N. Kartsonis
5Infectious Disease/Vaccines, Merck Research Laboratories, Upper Gwynedd, Pennsylvania 19454
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Mark T. Esser
1Wayne Clinical Support, Merck Research Laboratories, 466 Devon Park Dr., Wayne, Pennsylvania 19087-8630
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DOI: 10.1128/CVI.00478-08
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ABSTRACT

A direct binding Luminex assay has been developed and validated for the detection of human immunoglobulin G (IgG) antibodies to the Staphylococcus aureus iron surface determinant B protein (IsdB) in serum following natural infection or immunization with investigational Saccharomyces cerevisiae-derived IsdB-based vaccines. To ensure that IsdB-specific IgG antibodies are measured following immunization with S. cerevisiae-derived IsdB, an Escherichia coli-produced IsdB antigen is used in the assay. The IsdB antigen is covalently conjugated to maleimide microspheres via an engineered carboxy-terminal cysteine residue. Antibody titers are determined in a direct binding format, where the phycoerythrin-labeled monoclonal antibody (HP6043) specific for IgG1 to IgG4 binds to human serum IgG antibodies. Fluorescent signal emitted from bound HP6043 is directly proportional to an individual's antibody levels. A pooled human reference serum from vaccinees with high titers to IsdB is used to generate a 12-point standard curve. The correlation of mean fluorescent intensity (MFI) units to μg/ml of IsdB-specific IgG is made by interpolating the MFI data through a four-parameter curve-fitting algorithm. The assay is sensitive to 1.06 μg/ml with a dynamic range of 2.1 to 10,625 μg/ml. The overall specificity of the assay is >96% and the linearity (parallelism) of the assay is −4% per 10-fold dilution. The total precision of the assay was 16.6% relative standard deviation across three different IsdB antigen lots, three different microsphere lots, two secondary antibody lots, and three different operators. The assay has proven useful for evaluating the immune response following the administration of different dosages and formulations of investigational IsdB-based vaccines.

  • Copyright © 2009 American Society for Microbiology
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Serologic Assay To Quantify Human Immunoglobulin G Antibodies to the Staphylococcus aureus Iron Surface Determinant B Antigen
Michael D. Raedler, Samantha Heyne, Erica Wagner, Sheri K. Shalkowski, Susan Secore, Annaliesa S. Anderson, James Cook, Leslie Cope, Tessie McNeely, Mary Retzlaff, Jon Shanter, Leonard J. Rubinstein, Tina Green, N. Kartsonis, Mark T. Esser
Clinical and Vaccine Immunology May 2009, 16 (5) 739-748; DOI: 10.1128/CVI.00478-08

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Serologic Assay To Quantify Human Immunoglobulin G Antibodies to the Staphylococcus aureus Iron Surface Determinant B Antigen
Michael D. Raedler, Samantha Heyne, Erica Wagner, Sheri K. Shalkowski, Susan Secore, Annaliesa S. Anderson, James Cook, Leslie Cope, Tessie McNeely, Mary Retzlaff, Jon Shanter, Leonard J. Rubinstein, Tina Green, N. Kartsonis, Mark T. Esser
Clinical and Vaccine Immunology May 2009, 16 (5) 739-748; DOI: 10.1128/CVI.00478-08
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KEYWORDS

Antibodies, Bacterial
Cation Transport Proteins
Immunoglobulin G

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