1 a semi-automated method to detect opsonophagocytic killing (opk) janet c. onishi vaccine and...
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A semi-automated method to detectopsonophagocytic killing (OPK)
Janet C. OnishiVaccine and Biologics Research
Merck & Co. Inc.
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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.
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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
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OPK Assay Agar Plate Images (96 Samples)Samples applied using a 6 x 8 grid on 2 plates
Count colonies on microscope
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96 Well Filtration Millipore Plate OPK Assay with image analysis
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Transfer 10μL OPK reaction to 100μL H20 in HV 96 well plate
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Filtration System
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Filtering Method
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Incubate assay plates in humid environment
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Stain and Destain bacteria – Filtration to change solutions
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Stained assay plate
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Count colonies on image analyzer
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OPK Assay Readout - Millipore vs. Agar
Millipore Plate
Agar Plate
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Assay Template NIBSC serum samples tested against Pn 6B
380964 380828 380638 380808 380860 ControlsSerum dilution
1:64
1:128
1:256
1:512
1:1024
1:2048
1:4096
1:8192
QC 1:256
QC 1:512
QC 1:1024
QC1:256+6B Ps
QC 1:256+C-Ps
Medium only
Cells + C’No serum
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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
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Inter-Analyst Count Variability Results
Millipore Plate
Agar PlateMean
(95% CI)
ManualMean
(95% CI)
ComputerMean
(95% CI)
10.3%(5.8%, 42.2%)
14.3%(8%, 59.8%)
1.7%(0.9%, 6.7%)
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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.
OPK Assay Specificity - Image
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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