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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. - PowerPoint PPT Presentation
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Classical Classical opsonophagocytic killing opsonophagocytic killing
type assaytype assay
Sandra Romero-Steiner, Ph.D.Sandra Romero-Steiner, Ph.D.
Killing OPA AssayKilling OPA Assay
Romero-Steiner Romero-Steiner et alet al. CDLI . CDLI 1997;4:415-221997;4:415-22
Four componentsFour components SerumSerum BacteriaBacteria ComplementComplement Culturable phagocytesCulturable phagocytes
Internalized Pnc are killed Internalized Pnc are killed Viability as an endpointViability as an endpoint
Killing OPA assayKilling OPA assay
FcR CR1/CR3
+
PMN
C3b/iC3b
Ab
Bacteria
Viability as an Viability as an endpointendpoint
Assay Components - 1Assay Components - 1
SerumSerum 20 20 l (duplicate l (duplicate
wells)wells) InfantsInfants AdultsAdults ElderlyElderly High risk populationsHigh risk populations
SplenectomySplenectomy HIVHIV NavajoNavajo
Assay Components - 2Assay Components - 2 Bacteria Bacteria (CDLI, (CDLI,
1997)1997) Tp 4 Tp 4 (DS 2382-94)(DS 2382-94) Tp 6B Tp 6B (DS 2212-94)(DS 2212-94) Tp 9V Tp 9V (DS 400-92)(DS 400-92) Tp 14 Tp 14 (DS 2214-94)(DS 2214-94) Tp 18C Tp 18C (SP116)(SP116) Tp 19F Tp 19F (DS 2217-94)(DS 2217-94) Tp 23F Tp 23F (DS 2216-94)(DS 2216-94)
Tp 1, 3, 5, 7F, 12FTp 1, 3, 5, 7F, 12F
Assay components - 3Assay components - 3 Rabbit Complement 3-4 week, Rabbit Complement 3-4 week,
FrozenFrozen 31038-100BZ 100 ml Frozen, pooled, 31038-100BZ 100 ml Frozen, pooled,
sterile.sterile. 31038-1BZ 1 ml Frozen, pooled, sterile.31038-1BZ 1 ml Frozen, pooled, sterile.
Pel-Freez Clinical Systems, LLC Pel-Freez Clinical Systems, LLC Store Lyophilized powder at or below –20 Store Lyophilized powder at or below –20 ooCC Store Frozen liquid at or below –55 Store Frozen liquid at or below –55 ooCCDynal Biotech LLC.Dynal Biotech LLC.
9099 North Deerbrook Trail9099 North Deerbrook TrailBrown Deer, WI USA 53223Brown Deer, WI USA 53223Telephone: 1-800-558-4511Telephone: 1-800-558-4511Fax: 414-357-4518Fax: 414-357-4518e-mail: e-mail: [email protected]
Assay Components - 4Assay Components - 4
HL-60 cellsHL-60 cells Promyelocytic leukemia Promyelocytic leukemia ATCC, Rockville, MD ATCC, Rockville, MD CCL 240CCL 240 Passage 20-24Passage 20-24 Passage 130-160Passage 130-160 PMN-like differentiationPMN-like differentiation
100 mM DMF100 mM DMF 4 x 104 x 1055 cells/well cells/well
VIABILITY OPA
Unk1 Unk2 Unk3 Unk4 QC Unk1 Unk2 Unk3 Unk4 QC globulinglobulin
C’ controlsC’ controls
1:81:81:161:161:321:32 .. .. .. ..1:10241:1024
1:641:64 . . .. .. .. ..20482048
Romero-Steiner Romero-Steiner et al.et al. CDLI CDLI 2003;10:1019-242003;10:1019-24
Median OPA titersMedian OPA titers Five participating laboratoriesFive participating laboratories 24 sera (12 pre-post pairs)24 sera (12 pre-post pairs) AdultsAdults NIBSC NIBSC
Table 1. Median OPA titers for QC Table 1. Median OPA titers for QC serasera
SerumSerum 44 6B6B 9V9V 1414 18C18C 19F19F 23F23F
380329a 4 4 4 64 4 4 4
381039b 16 4 32 256 8 64 8
380318 4 4 4 4 4 16 4
380773 256 16 8 2048 4 128 4
380342 4 4 4 4 4 4 4
381399 4 4 4 4 8 4 4
380292 4 4 4 16 4 4 4
380807 64 4 128 4096 128 16 8
380376 4 4 4 4 4 4 4
380847 4 4 4 1024 32 4 4
380372 4 128 32 8 4 4 4
380964 32 128 128 1024 32 16 4
380360 64 64 4 64 4 4 4
380828 128 64 256* 32* 256 64 4
380330 4 16 4 4 4 16 4
380638 32 64 64 4 64 256 8
380386 4 4 4 4 4 4 4
380859 4 16 64 512 4 16 4
380327 4 4 4 4 4 4 4
380808 64 4 64 256 128 128 128
380351 4 4 4 4 64 4 4
380824 2048 64 64 512 64 16 1024
380298 4 4 4 512 8 4 4
380860 128 256 128 2048 256 512 128
Table 2. Percentage of sera within 1 and 2 Table 2. Percentage of sera within 1 and 2 dilutions about the median OPA titerdilutions about the median OPA titer
Multi-laboratory Evaluation Multi-laboratory Evaluation
S. S. pneumoniapneumonia
eeSerotypeSerotype
Lab 1 Lab 2 Lab 3 Lab 4 Lab Lab 1 Lab 2 Lab 3 Lab 4 Lab 55
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 83, 88 75, 94 83, 96 83, 92
14 63, 83 75, 83 81, 94 92, 92 88, 92
18C 88, 96 88, 96 56, 69 88, 92 88, 100
19F 79, 96 88, 96 94, 100 96, 100 88, 100
23F 100, 100 100, 100
100, 100 96, 100 92, 96
a Within one dilution about the median OPA titer – 75% overall b Within two dilutions about the median OPA titer – 88% overall
Log
2 O
PA
Tit
er
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Lab 1Lab 2Lab 3Lab 4Lab 5
Type 4 A38
0329
3810
3938
0318
3807
7338
0342
3813
9938
0292
3808
0738
0376
3808
4738
0372
3809
6438
0360
3808
2838
0330
3806
3838
0386
3808
5938
0327
3808
0838
0351
3808
2438
0298
3808
60
Log
2 O
PA
Tit
er
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Type 14 D
Serum Sample
3803
2938
1039
3803
1838
0773
3803
4238
1399
3802
9238
0807
3803
7638
0847
3803
7238
0964
3803
6038
0828
3803
3038
0638
3803
8638
0859
3803
2738
0808
3803
5138
0824
3802
9838
0860
Log
2 O
PA
Tit
er
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Type 23F G
CDLI 2003; 10: 1021, Figure 1CDLI 2003; 10: 1021, Figure 1
Higher agreement Higher agreement in sera with low in sera with low titerstiters Lower agreement Lower agreement in sera with high in sera with high titerstiters
Multi-laboratory EvaluationMulti-laboratory Evaluation
S. pneumoniae serotype
Agr
eem
ent
of O
PA
Tit
ers
(mea
n +
95%
CI)
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5
4 6B 9V 14 18C 19F 23F
Figure 2.
Log2 ELISA concentration (g/ml)
-2 0 2 4 6 8 10
Log
2 O
PA
tit
er
0
2
4
6
8
10
12
14
Type 4Type 6BType 9VType 14Type 18CType 19FType 23FLinear regression
r = 0.79slope = 1.2n = 49
Figure 3.
Validation of OPAValidation of OPA
B. T. Hu et al. CDLI 2005, 12: 287-295.B. T. Hu et al. CDLI 2005, 12: 287-295. Specificity for 9 serotypes (1 & 5)Specificity for 9 serotypes (1 & 5)
Homologous Ps Homologous Ps >> 80% 80% ((>> 87% CDLI, 1997) 87% CDLI, 1997) Heterologous Ps Heterologous Ps << 20% 20%
Intermediate PrecisionIntermediate Precision 4 serum specimens over 6 months (n=20-30)4 serum specimens over 6 months (n=20-30) Panel of infant sera on 3 days and 3 Panel of infant sera on 3 days and 3
operatorsoperators Overall 81 % of titers within 2 dil. of medianOverall 81 % of titers within 2 dil. of median
Validation Cont.Validation Cont.
Linearity (9 serotypes)Linearity (9 serotypes) 2 sera at 4 initial dilutions with PMNs 400:12 sera at 4 initial dilutions with PMNs 400:1 r = 0.982 to 1.000, slopes = -0.850 to -1.350r = 0.982 to 1.000, slopes = -0.850 to -1.350
Accuracy (9 serotypes)Accuracy (9 serotypes) Negative serum spiked with Positive serumNegative serum spiked with Positive serum Agreement of Obs / Exp for 2 seraAgreement of Obs / Exp for 2 sera All types 100% except 14 (81%) & 23F (75%)All types 100% except 14 (81%) & 23F (75%)
Validation Cont.Validation Cont.
RobustnessRobustness Bacterial strains (CDLI, 1997;4:420 – Fig 3.)Bacterial strains (CDLI, 1997;4:420 – Fig 3.) Exogenous C’ (Exogenous C’ (OpaqueOpaque/Transparent)/Transparent) Shaking period (Opsonization/Shaking period (Opsonization/PhagocytosisPhagocytosis)) Effector:target cell ratio (Effector:target cell ratio (400:1400:1, 100:1, 50:1), 100:1, 50:1) HL-60 cell passage # (HL-60 cell passage # (<37<37, <80, 111, 160), <80, 111, 160)
Advantages Advantages DisadvantagesDisadvantages
Reference methodReference method www.vaccine.uab.ewww.vaccine.uab.e
dudu StandardizedStandardized ValidatedValidated Culturable Culturable
phagocytesphagocytes Endpoint = Viability Endpoint = Viability Technology transferTechnology transfer
Single serotypeSingle serotype Volume of seraVolume of sera Non-automatedNon-automated Colony countingColony counting Discontinuous Discontinuous
titerstiters
vOPA fOPA
ASM 105ASM 105thth general generalMeeting - June 6Meeting - June 6thth
Poster V-001, Poster V-001, Board 545,Board 545,
9am - 12 noon9am - 12 noon