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: uscustserv@dynalbiotech.com

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