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Classical opsonophagocytic killing type assay

Classical opsonophagocytic killing type assay. Sandra Romero-Steiner, Ph.D. Killing OPA Assay. Romero-Steiner et al . CDLI 1997;4:415-22 Four components Serum Bacteria Complement Culturable phagocytes Internalized Pnc are killed Viability as an endpoint. CR1/CR3. Fc g R. Bacteria.

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Classical opsonophagocytic killing type assay

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  1. Classical opsonophagocytic killing type assay Sandra Romero-Steiner, Ph.D.

  2. Killing OPA Assay • Romero-Steiner et al. CDLI 1997;4:415-22 • Four components • Serum • Bacteria • Complement • Culturable phagocytes • Internalized Pnc are killed • Viability as an endpoint

  3. CR1/CR3 Fcg R Bacteria Ab + C3b/iC3b PMN Killing OPA assay Viability as an endpoint

  4. Assay Components - 1 • Serum • 20 ml (duplicate wells) • Infants • Adults • Elderly • High risk populations • Splenectomy • HIV • Navajo

  5. Assay Components - 2 • Bacteria (CDLI, 1997) • Tp 4 (DS 2382-94) • Tp 6B (DS 2212-94) • Tp 9V (DS 400-92) • Tp 14 (DS 2214-94) • Tp 18C (SP116) • Tp 19F (DS 2217-94) • Tp 23F (DS 2216-94) • Tp 1, 3, 5, 7F, 12F

  6. Assay components - 3 • Rabbit Complement 3-4 week, Frozen • 31038-100BZ 100 ml Frozen, pooled, sterile. • 31038-1BZ 1 ml Frozen, pooled, sterile. • Pel-Freez Clinical Systems, LLC • Store Lyophilized powder at or below –20 oC • Store Frozen liquid at or below –55 oC Dynal Biotech LLC.9099 North Deerbrook TrailBrown Deer, WI USA 53223Telephone: 1-800-558-4511Fax: 414-357-4518e-mail: uscustserv@dynalbiotech.com

  7. Assay Components - 4 • HL-60 cells • Promyelocytic leukemia • ATCC, Rockville, MD • CCL 240 • Passage 20-24 • Passage 130-160 • PMN-like differentiation • 100 mM DMF • 4 x 105 cells/well

  8. Unk1 Unk2 Unk3 Unk4 QC gglobulin 1:8 1:16 1:32 . . . . 1:1024 1:64 . . . . . 2048 C’ controls VIABILITY OPA

  9. Table 1. Median OPA titers for QC sera • Romero-Steiner et al. CDLI 2003;10:1019-24 • Median OPA titers • Five participating laboratories • 24 sera (12 pre-post pairs) • Adults • NIBSC

  10. Table 2. Percentage of sera within 1 and 2 dilutions about the median OPA titer Multi-laboratory Evaluation a Within one dilution about the median OPA titer – 75% overall b Within two dilutions about the median OPA titer – 88% overall

  11. Multi-laboratory Evaluation • Higher agreement in sera with low titers • Lower agreement in sera with high titers CDLI 2003; 10: 1021, Figure 1

  12. Validation of OPA • B. T. Hu et al. CDLI 2005, 12: 287-295. • Specificity for 9 serotypes (1 & 5) • Homologous Ps > 80% (> 87% CDLI, 1997) • Heterologous Ps < 20% • Intermediate Precision • 4 serum specimens over 6 months (n=20-30) • Panel of infant sera on 3 days and 3 operators • Overall 81 % of titers within 2 dil. of median

  13. Validation Cont. • Linearity (9 serotypes) • 2 sera at 4 initial dilutions with PMNs 400:1 • r = 0.982 to 1.000, slopes = -0.850 to -1.350 • Accuracy (9 serotypes) • Negative serum spiked with Positive serum • Agreement of Obs / Exp for 2 sera • All types 100% except 14 (81%) & 23F (75%)

  14. Validation Cont. • Robustness • Bacterial strains (CDLI, 1997;4:420 – Fig 3.) • Exogenous C’ (Opaque/Transparent) • Shaking period (Opsonization/Phagocytosis) • Effector:target cell ratio (400:1, 100:1, 50:1) • HL-60 cell passage # (<37, <80, 111, 160)

  15. Reference method www.vaccine.uab.edu Standardized Validated Culturable phagocytes Endpoint = Viability Technology transfer Single serotype Volume of sera Non-automated Colony counting Discontinuous titers Advantages Disadvantages

  16. ASM 105th general Meeting - June 6th Poster V-001, Board 545, 9am - 12 noon fOPA vOPA

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