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

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

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

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

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 Trail Brown Deer, WI USA 53223 Telephone: 1-800-558-4511 Fax: 414-357-4518 e-mail: uscustserv@dynalbiotech.com

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

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

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

Serum 4 6B 9V 14 18C 19F 23F 380329a 64 381039b 16 32 256 8 380318 380773 2048 128 380342 381399 380292 380807 4096 380376 380847 1024 380372 380964 380360 380828 256* 32* 380330 380638 380386 380859 512 380327 380808 380351 380824 380298 380860

Table 2. Percentage of sera within 1 and 2 dilutions about the median OPA titer Multi-laboratory Evaluation S. pneumoniae Serotype Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 4 88a, 100b 79, 88 100, 100 96, 96 96, 100 6B 75, 88 83, 100 56, 72 83, 88 88, 92 9V 63, 71 75, 94 83, 96 83, 92 14 63, 83 75, 83 81, 94 92, 92 18C 88, 96 56, 69 88, 100 19F 79, 96 94, 100 23F 92, 96 a Within one dilution about the median OPA titer – 75% overall b Within two dilutions about the median OPA titer – 88% overall

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

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

Validation Cont. Linearity (9 serotypes) Accuracy (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%)

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)

Advantages Disadvantages 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

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