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. Killing OPA assay Viability as an endpoint + Fcg R CR1/CR3 PMN
C3b/iC3b Ab
Bacteria
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 )
Tp 9V (DS )
Tp (DS )
Tp 18C (SP116)
Tp 19F (DS )
Tp 23F (DS )
Tp 1, 3, 5, 7F, 12F

6. Assay components - 3 Rabbit Complement 3-4 week, Frozen
BZ 100 ml Frozen, pooled, sterile.
BZ 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 North Deerbrook Trail Brown Deer, WI USA Telephone: Fax:

7. Assay Components - 4 HL-60 cells Promyelocytic leukemia
ATCC, Rockville, MD
CCL 240
Passage 20-24
Passage
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:
Median OPA titers
Five participating laboratories
24 sera (12 pre-post pairs)
Adults
NIBSC

10. 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

11. 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

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

15. 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

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

17. 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)

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

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