Classical opsonophagocytic killing type assay

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Classical opsonophagocytic killing type assay

• Sandra Romero-Steiner, Ph.D.

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Killing OPA Assay

• Romero-Steiner et al. CDLI 19974415-22
• Four components
• Serum
• Bacteria
• Complement
• Culturable phagocytes
• Internalized Pnc are killed
• Viability as an endpoint

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Killing OPA assay
Viability as an endpoint
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Assay Components - 1

• Serum
• 20 ml (duplicate wells)
• Infants
• Adults
• Elderly
• High risk populations
• Splenectomy
• HIV
• Navajo

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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

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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 [phone]e-mail
uscustserv_at_dynalbiotech.com
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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

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Unk1 Unk2 Unk3 Unk4 QC gglobulin
18 116 132 . . . . 11024
164 . . . . . 2048
C controls
VIABILITY OPA
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Table 1. Median OPA titers for QC sera

• Romero-Steiner et al. CDLI 2003101019-24
• Median OPA titers
• Five participating laboratories
• 24 sera (12 pre-post pairs)
• Adults
• NIBSC

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Serum 4 6B 9V 14 18C 19F 23F
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
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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 Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 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 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
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Multi-laboratory Evaluation

• Higher agreement in sera with low titers
• Lower agreement in sera with high titers

CDLI 2003 10 1021, Figure 1
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Validation of OPA

• B. T. Hu et al. CDLI 2005, 12 287-295.
• Specificity for 9 serotypes (1 5)
• Homologous Ps gt 80 (gt 87 CDLI, 1997)
• Heterologous Ps lt 20
• Intermediate Precision
• 4 serum specimens over 6 months (n20-30)
• Panel of infant sera on 3 days and 3 operators
• Overall 81 of titers within 2 dil. of median

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Validation Cont.

• Linearity (9 serotypes)
• 2 sera at 4 initial dilutions with PMNs 4001
• 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)

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Validation Cont.

• Robustness
• Bacterial strains (CDLI, 19974420 Fig 3.)
• Exogenous C (Opaque/Transparent)
• Shaking period (Opsonization/Phagocytosis)
• Effectortarget cell ratio (4001, 1001, 501)
• HL-60 cell passage (lt37, lt80, 111, 160)

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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

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