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,

3 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

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 H 2 0 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 Controls Serum 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

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 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%)

17 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

18 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