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Flow Cytometric Opsonophagocytic Assays

Flow Cytometric Opsonophagocytic Assays. Joseph E. Martinez CDC, Atlanta. S. pneumoniae. Multiplexed OPA work supported in part by a non-restricted CRADA with Flow Applications, Inc. and covered under US patent 6,815,172. Opsonophagocytic Assays (OPA ) Two approaches for OP measurement

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Flow Cytometric Opsonophagocytic Assays

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  1. Flow Cytometric Opsonophagocytic Assays Joseph E. Martinez CDC, Atlanta S.pneumoniae Multiplexed OPA work supported in part by a non-restricted CRADA with Flow Applications, Inc. and covered under US patent 6,815,172

  2. Opsonophagocytic Assays (OPA) • Two approaches for OP measurement • - Killing assays (Steiner, et al., others) • - Uptake assays (Martinez; Jansen)

  3. Opsonophagocytic Assay

  4. FALS Sensor Fluorescence Fluorescence detector (PMT3, PMT4 etc.)

  5. Flow Opsonophagocytic Assays Phagocytic cells Complement Ctl Positive Rx OPA Graph

  6. Bacteria or Microspheres S. pneumoniae Microspheres

  7. Multiplexed Flow OPA Single-plex Multiplex

  8. Correlations between reference OPA and flow OPA 1Different strains used

  9. Technical Considerations • Targets • 1. Bacteria • -Variable label

  10. Variability of Labeled Bacteria Positive Cells Negative Cells

  11. Technical Considerations • Targets • 1. Bacteria • -Variable label • 2. Beads • -Standardized label

  12. Beads Provided a Standard Fluorescent Target CDC, Sero 4-Y Beads FA, Sero 4-Y Beads

  13. Technical Considerations • Effector Cells • 1. Donor PMNs • 2. HL60 PMNs

  14. Comparison of PMN and HL60 PMN Derived OPA Titers

  15. Technical Considerations Cont. • Effector Cells • 1. Donor PMNs • 2. HL60 PMNs • a. Culture maintenance

  16. Effects of Different Culture Conditions 38% S and G2/M 16% S and G2/M 3% S and G2/M Published Induction Protocol Modified Induction Protocol HL60 Stock 3% 18% 57%

  17. Technical Considerations • Effector Cells • 1. Donor PMNs • 2. HL60 PMNs • a. Culture maintenance • b. Induction protocol

  18. Effects of Culture Conditions on HL60 Differentiation and Function Normal Culture Method Roller Culture Method

  19. Data Collection • Gating • Minimize overlap of “dead” cells within gate • Ensure gate contains a cells with ingested targets

  20. Gating Effects

  21. Data Collection • Gating • Minimize overlap of “dead” cells within gate • Ensure gate contains a cells with ingested targets • Compensation • Critical in multiplexed assays

  22. Data Analysis-Automation • Flow cytometric OPA can be automated • Sample handler • Batch file analysis of raw data files • Attractors • FlowJo • Curve fitting software to determine titer • Statlia • GLP compatible

  23. Automation Efficiencies

  24. Assay Comparisons *Overnight incubation required

  25. Advantages and Disadvantages

  26. Flow Cytometric OPA Technology • Can be applied to other bacteria cleared through an OP mechanism • S. aureus (Vernachio) Figure 1. Opsonization of ClfA-coated fluorescent beads. PMNs were incubated with unopsonized beads , complement opsonized beads, T1-2 plus complement opsonized beads, and non-specific human IgG1 complement opsonized beads. The phagocytic product (PP) represents the mean beads per cell multiplied by the percent fluorescent PMNs, as determined via flow cytometric analysis.

  27. Summary • Non-infectious targets • Correlate well with reference OP method • High throughput • Automation can further increase relative throughputs • Meet GLP guidelines

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