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A semi-automated method to detect opsonophagocytic killing (OPK) Janet C. Onishi

A semi-automated method to detect opsonophagocytic killing (OPK) Janet C. Onishi Vaccine and Biologics Research Merck & Co. Inc. Contributors. Michael Caulfield Su Wang Xu Liu Mayur Shah Lisa Sendi Angela Payne. Joe Antonello Greg Golm.

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A semi-automated method to detect opsonophagocytic killing (OPK) Janet C. Onishi

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  1. A semi-automated method to detect opsonophagocytic killing (OPK) Janet C. Onishi Vaccine and Biologics Research Merck & Co. Inc.

  2. Contributors • Michael Caulfield • Su Wang • Xu Liu • Mayur Shah • Lisa Sendi • Angela Payne • Joe Antonello • Greg Golm Liu, X., Wang, S., Sendi, L. and Caulfield, M.J. (2004) High-throughput imaging of bacterial colonies grown on filter plates with application to serum bactericidal assays. J Immunol Methods 292, 187-93.

  3. OPK Assay SummaryBasic assay design – S. Romero-Steiner • Opsonization • Perform serial dilutions of serum • Add bacteria • Add complement • Phagocytosis • Add differentiated myelocytic cells (HL-60 cell line) • Killing • Plate residual bacteria & count colonies • Determine endpoint titers

  4. OPK Assay Agar Plate Images (96 Samples)Samples applied using a 6 x 8 grid on 2 plates Count colonies on microscope

  5. 96 Well Filtration Millipore Plate OPK Assay with image analysis

  6. Transfer 10μL OPK reaction to 100μL H20 in HV 96 well plate

  7. Filtration System

  8. Filtering Method

  9. Incubate assay plates in humid environment

  10. Stain and Destain bacteria – Filtration to change solutions

  11. Stained assay plate

  12. Count colonies on image analyzer

  13. OPK Assay Readout - Millipore vs. Agar Millipore Plate Agar Plate

  14. Assay Template NIBSC serum samples tested against Pn 6B Serum dilution 1:64 1:128 1:256 1:512 1:1024 1:2048 1:4096 1:8192 380964 380828 380638 380808 380860 Controls Cells + C’ No serum QC 1:256 QC 1:512 QC 1:1024 QC1:256+6B Ps QC 1:256+C-Ps Medium only

  15. OPK Assay Analytical Validation Parameters • Count Variability (Analysts and Machine) • Relationship between CFU Level and Volume Plated • Comparison of CFU Levels Between Periphery and Central Wells • Extra-Variability Specification (for replicate samples) • OPK Titer Determination • Ruggedness (Analyst, Passage, Plate, Counting Method) • Assay Precision • Sero-status Cutoff • Specificity • Assay QC

  16. Inter-Analyst Count Variability Results

  17. OPK Assay Specificity - Image No Antiserum (Control) Antisera (1:64) Antisera + PnPs 23F Antisera + C-Ps * Note: all wells contained HL60 cells, serotype 23F bacteria, and baby rabbit complement.

  18. Summary of OPK Analytical ValidationStudies • All ruggedness parameters (operators, cell passages, plates and counting methods) within ~two-fold • Negative control samples (>95%) are below the limit of detection (LOD) • Supports seroconversion cut-off of 1:8 • New Merck assay comparable to CDC agar plate method • Higher throughput and less variable

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