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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