2-fold/4-fold multiplexed opsonophagocytic killing assay (MOPA) Nahm/Burton UAB Birmingham, AL 6/5/05

1
2-fold/4-fold multiplexed opsonophagocytic
killing assay (MOPA)Nahm/BurtonUABBirmingham,
AL6/5/05
2
How can we do the killing assay faster?
Classical OPA 0.6 plates/day
???
6 mph
60 mph
600 mph
3
Classical opsonophagocytic killing
assaySituation in 1990s

• Improvements made
• Use of microtiter plates
• Use of aliquots of frozen bacteria
• Use of HL-60 cell line as phagocytes
• Improvements needed
• Counting colonies is slow. ? Automate Counting
• Need much sera and reagents ? Multiplex the assay

4
(No Transcript)
5
Stained pneumococcal colonies
Colony diameter 0.1- 0.3 mm
Target area 8 mm x 25 mm
TTC Dye
6
(No Transcript)
7
(No Transcript)
8
Counting Speed Comparison

• Counting 3 microtiter plates (96x3 wells)
• Manual counting ? 144 min. (30 sec per well)
• Machine counting ? 10 min. (2 sec per well)
• Reading 3 ELISA plates
• 3-6 min. (1-2 min. per plate).

9
How can we do the killing assay faster?
Classical OPA 0.6 plates/day
OPA with auto. counting 6 plates/day
???
6 mph
60 mph
600 mph
10
Improve OPKA - Multiplex OPKA (double)
19F
Optochin Plate
6B
Strep. Plate
11
Multiplex OPKAVery Similar to the Classic OPKA.

• Add bacteria mixture (10 ul) antiserum (20 ul)
• Incubate (30 min, RT)
• Add complement (10 ul) phagocytes (40 ul)
• Incubate (60 min, 37oC, shake)
• Plate the reaction mixture on several agar
  plates.
• Pour an overlay agar with TTC and antibiotics.
• Incubate overnight.
• Count colonies.

12
Advantages of the multiplex assay

• Conserve serum from children
• Conserve other reagents
• Reduce overall effort
• The principle can be applied to other functional
  assays (e.g., bactericidal assay)

13
9 pneumococcal target strains
Serotype 4 6A 6B 9V 14 18C 19A 19F 23F
Name ORP4 ORP6A STRP6B STRP9V ORP14 ORP18C STRP 19A ORP 19F STRP23F
14
Comparison of single- vs double- serotype OPKA
(4 9V)


log(double)0.98log(single)0.04, R2 0.98
log(double)0.92log(single)0.11, R2 0.93,
2-fold outlier3
2-fold outlier11
15
Comparison of single- vs double- serotype OPKA
(6B 19F)


log(double)0.94log(single)0.01, R20.92
log(double)0.94log(single)0.05,
R20.97
2-fold outlier12
2-fold outlier3
16
Comparison of single- vs double- serotype OPKA
(18C 23F)


log(double)0.96log(single)0.08, R20.97
log(double)0.93log(single)0.06, R20.98
2-fold outlier3
2-fold outlier4
17
MOPA-4
18
Why MOPA-4?
19
MOPA-4 Protocol Summary
SOPA
MOPA
Add 20 ul Serum and 10 ul Bacteria (1000
cfu/well, 1 serotype)
Add 20 ul Serum and 10 ul Bacteria (500
cfu/well each serotype, 4 serotypes)
30 minutes, room temperature, no shaking
Opsonization Phase
Add 10 ul Complement and 40 ul HL60 (ET4001)
Add 10 ul Complement and 40 ul HL60 (ET2001)
45 minutes, 37C, shaking
Phagocytosis Phase
Spot 5 ul to single plate, add overlay with TTC
Spot 10 ul to four replicate plates, add overlay
with TTC and antibiotic (streptomycin,
spectinomycin, trimethoprim, optochin, or
rifampicin)
Incubate overnight, count colonies.
20
Bacteria selection

• Serotype
• Antibiotic sensitivity/resistivity
• Derive strains resistant to spectinomycin,
  streptomycin, trimethoprim, or optochin, but
  sensitive to other common antibiotics.

• Phase variation
• All strains presented were gt95 opaque, except
  OREP18C which was gt50 transparent.
• 4. Performance in OPKA (SOPA format)
• a. Killed only by appropriate hybridoma.
• b. Low background killing.
• c. Again checked for resistance/sensitivity to
  appropriate antibiotic.

21
Bacteria strain antibiotic sensitivity/resistivity

22
Proposed MOPA-4 groups
Group A Group B Group C Group D
Optochin OREP4 OREP18C OREP7F  
Spectinomycin SPECREP6B SPECREP19F SPECREP1  
Streptomycin STREP14 EMC9V STREP5  
Trimethoprim TRIREP19A EMC23F TRIREP6A  
Rifampicin       RIFREP3
Note 1. 7-valent PS vaccine serotypes covered
by Groups A and B. 2. Serotypes 6A and 19A may
be interchanged, as desired.
23
SOPA vs MOPA-4
R20.98
R20.95 (R20.94)
MOPA-4
SOPA
24
SOPA vs MOPA
R20.98
R20.60 (R20.99)
MOPA-4
SOPA
25
How can we do the killing assay faster?
Classic OPA 0.6 plates/day
OPA with colony counting 6 plates/day
MOPA-4 60 plates/day?
6 mph
60 mph
600 mph
26
(No Transcript)
27
Acknowledgements Moon Nahm Lab
Bill Benjamin
Kyung Hyo (Kay) Kim
Jigui Yu
Jisheng Lin
Jong Taek Kim
Linda Savage
Michael Putman
David Briles Lab Janice King
28
ThankYou