Transcript ASM - OPK - University of Alabama at Birmingham

A semi-automated method to detect
opsonophagocytic killing (OPK)
Janet C. Onishi
Vaccine and Biologics Research
Merck & Co. Inc.
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Contributors
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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 Summary
Basic 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
Serum
dilution
1:64
1:128
380964
380828
380638
380808 380860
Controls
Cells + C’
No serum
1:256
QC 1:256
1:512
QC 1:512
1:1024
QC 1:1024
1:2048
QC1:256+6B Ps
1:4096
1:8192
QC 1:256+C-Ps
Medium only
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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 Plate
Mean
(95% CI)
Manual
Mean
(95% CI)
Computer
Mean
(95% CI)
10.3%
(5.8%, 42.2%)
14.3%
(8%, 59.8%)
1.7%
(0.9%, 6.7%)
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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.
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Summary of OPK Analytical Validation
Studies
• 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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