1. 2-fold/4-fold multiplexed opsonophagocytic killing assay (MOPA) Nahm/Burton UAB Birmingham, AL 6/5/05

2. 6 mph 60 mph600 mph Classical OPA 0.6 plates/day How can we do the killing assay faster? ???

3. Classical opsonophagocytic killing assay Situation 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

5. TTC Dye Stained pneumococcal colonies Colony diameter 0.1- 0.3 mm Target area 8 mm x 25 mm

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. 6 mph 60 mph600 mph Classical OPA 0.6 plates/day How can we do the killing assay faster? ??? OPA with auto. counting 6 plates/day

10. Improve OPKA - Multiplex OPKA (double) Optochin Plate Strep. Plate 19F 6B

11. Multiplex OPKA Very 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, 37 o C, 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 Serotype46A6B9V1418C19A19F23F NameORP 4 ORP 6A STRP 6B STRP 9V ORP 14 ORP 18C STRP 19A ORP 19F STRP 23F

14. Comparison of single- vs double- serotype OPKA (4 & 9V) log(double)=0.98[log(single)]+0.04, R 2 = 0.98log(double)=0.92[log(single)]+0.11, R 2 = 0.93, 2-fold outlier;32-fold outlier;11

15. log(double)=0.94[log(single)]+0.01, R 2 =0.92 log(double)=0.94[log(single)]+0.05, R 2 =0.97 Comparison of single- vs double- serotype OPKA (6B & 19F) 2-fold outlier;122-fold outlier;3

16. log(double)=0.96[log(single)]+0.08, R 2 =0.97 log(double)=0.93[log(single)]+0.06, R 2 =0.98 Comparison of single- vs double- serotype OPKA (18C & 23F) 2-fold outlier;32-fold outlier;4

17. MOPA-4

18. Why MOPA-4?

19. MOPA-4 Protocol Summary Opsonization Phase SOPAMOPA 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 Add 10 ul Complement and 40 ul HL60 (E:T~400:1) Add 10 ul Complement and 40 ul HL60 (E:T~200:1) Phagocytosis Phase 45 minutes, 37°C, shaking 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: 1.Serotype 2.Antibiotic sensitivity/resistivity Derive strains resistant to spectinomycin, streptomycin, trimethoprim, or optochin, but sensitive to other common antibiotics. 3.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.

21. Bacteria strain antibiotic sensitivity/resistivity

22. Group AGroup BGroup CGroup D OptochinOREP4OREP18COREP7F SpectinomycinSPECREP6BSPECREP19FSPECREP1 StreptomycinSTREP14EMC9VSTREP5 TrimethoprimTRIREP19AEMC23FTRIREP6A Rifampicin RIFREP3 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.

23. SOPA vs MOPA-4 R 2 =0.95 (R 2 =0.94) R 2 =0.98 SOPA MOPA-4

24. SOPA vs MOPA R 2 =0.98R 2 =0.60 (R 2 =0.99) SOPA MOPA-4

25. 6 mph 60 mph 600 mph Classic OPA 0.6 plates/day OPA with colony counting 6 plates/day MOPA-4 60 plates/day? How can we do the killing assay faster?

27. Acknowledgements Moon Nahm Lab Kyung Hyo (Kay) Kim Jigui Yu Jisheng Lin Jong Taek Kim Linda Savage David Briles Lab Janice King Bill Benjamin Michael Putman

28. Thank You