2-fold/4-fold multiplexed opsonophagocytic killing assay (MOPA)Nahm/BurtonUABBirmingham, AL6/5/05
How can we do the killing assay faster? Classical OPA 0.6 plates/day ??? 6 mph 60 mph 600 mph
Classical opsonophagocytic killing assaySituation in 1990’s • 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
Stained pneumococcal colonies Colony diameter 0.1- 0.3 mm Target area 8 mm x 25 mm TTC Dye
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).
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
Improve OPKA - Multiplex OPKA (double) 19F Optochin Plate 6B Strep. Plate
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.
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)
Comparison of single- vs double- serotypeOPKA (4 & 9V) log(double)=0.98[log(single)]+0.04, R2 = 0.98 log(double)=0.92[log(single)]+0.11, R2 = 0.93, 2-fold outlier;3 2-fold outlier;11
Comparison of single- vs double- serotypeOPKA (6B & 19F) log(double)=0.94[log(single)]+0.01, R2=0.92 log(double)=0.94[log(single)]+0.05, R2=0.97 2-fold outlier;12 2-fold outlier;3
Comparison of single- vs double- serotypeOPKA (18C & 23F) log(double)=0.96[log(single)]+0.08, R2=0.97 log(double)=0.93[log(single)]+0.06, R2=0.98 2-fold outlier;3 2-fold outlier;4
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 OpsonizationPhase Add 10 ul Complement and 40 ul HL60 (E:T~400:1) Add 10 ul Complement and 40 ul HL60 (E:T~200:1) 45 minutes, 37°C, 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.
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 >95% opaque, except OREP18C which was >50% 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.
Proposed MOPA-4 groups Note: 1. 7-valent PS vaccine serotypes covered by Groups A and B. 2. Serotypes 6A and 19A may be interchanged, as desired.
SOPA vs MOPA-4 R2=0.98 R2=0.95 (R2=0.94) MOPA-4 SOPA
SOPA vs MOPA R2=0.98 R2=0.60 (R2=0.99) MOPA-4 SOPA
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
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