In vitro quantitation of Theileria parva sporozoites for use - TropMed ...

158 Appendices
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APPENDIX VII
Sporozoite Neutralization Assay protocol
Day -1: PBMC isolation
Prepare PBMC at 6*10 6 /ml as describes in Appendix I
Aliquot in 2 flat-bottomed Nunclon ® microtitration plates (Nunclon, Roskilde) (40 µl /well)
Store at 37°C in a CO 2 incubator
Store the rest of PBMC in culture bottles in the refrigerator
Day 0: In vitro infection
PLATE SETUP (an example comparing pre- and post-immune sera from three animals is given)
Label the titration plates according to the test sera:
Plate 1, three rows each for 131 pre, 6146 pre, 6148 pre and FCS.
Plate 2, three rows each for 6146 post, 6148 post, 131 post and p67.
IN VITRO TITRATION
Thaw stabilate diluent (15 ml) in a 37°C water bath
Thaw 100 µl of each test serum
Transfer 320 µl of refrigerator PBMC to each of eight Eppendorf tubes (labelled for each
serum)
Add 80 µl of test sera to each PBMC tube and suspend in a vortex mixer for a few seconds
Transfer 10 µl of PBMC/serum mixture to each well of respective rows. Use separate tips for
each PBMC/serum mix
Place plates at 37°C in a CO 2 incubator
Thaw 5 ml of T. parva stabilate in a water bath (37°C) for 5 min (record time)
Transfer stabilate to a 50 ml Falcon ® tube, merging the vials
Centrifuge at 400 g for 10 min
Recover supernatants in a sterile tube using a pastet (dilution 1)
Prepare seven dilution tubes with 2 ml of diluent in each
Make eight serial dilutions of stabilate (2 ml stabilate in 2 ml diluent) (dilutions 2 to 8)