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

Chapter 8: SNA and refinement of titration protocol 105
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scatter (FSC) versus side scatter (SSC) constructed. FSC measures size of cells while SSC the
complexity of intracellular contents.
On another occasion, PBMC were collected and portion of the suspension distributed to 96-well
microtitration plates (Nunc ® , Roskilde, Denmark) at 40 µl/well. The 96-well plates were incubated
overnight at 37°C in an incubator (5% CO 2 in air). The remaining portion was stored in a 50 ml
Cellstar ® tube (Greiner Bio-One, Frickenhausen, Germany) in a refrigerator. This set was for
demonstrating sporozoite neutralisation.
8.2.2. Exclusion of Con-A
A 96-well culture plate of T. parva-infected lymphoblasts was prepared as described in Appendix
I.A with the activation step excluded. Another plate was set up with the original protocol (Appendix
I.A without alterations) as a control. Cyto-centrifuge smears were made after 10 days of incubation
and compared.
8.2.3. Use of 96-wellmicrotitration plates Vs microtubes
Two titrations, one with the original (Appendix I.A) and the other with the refined (Appendix I.B)
protocols were conducted using glycerol and sucrose stabilates to assess the effect of using plates
for dilutions and incubation. The PBMC were collected, isolated and stored as described in
Appendix I.B. The titrations were done the following day and cultures scored from cyto-centrifuge
smears after 10 days. The refined protocol included a 1 minute shaking of plates after 30 minutes of
primary incubation instead of the 1 hour shaking in microtubes for the original protocol. Effects of
this reduced period of shaking on the infectivity of stabilates were analysed.
8.2.4. Use of PCR for reading titration results
A titration of glycerol, sucrose and trehalose stabilates was set up as described in chapter 5. After
removal of samples for cyto-centrifuge smears (100 µl from each well), the plates were treated as
described in Appendix VI for DNA extraction and PCR reading. The PCR products were then run
though 2 % (w/v) Agarose gel. Each well tested on PCR was scored positive or negative and
compared to the corresponding result from Giemsa-stained smears (Figure 19).
Analysis of the agreement between the two scoring procedures was done by calculating Cohen's
kappa and McNemar's chi-square. Landis & Koch (1977) proposed the following scale to describe
the degree of agreement between tests: 0.21-0.40, "Fair"; 0.41-0.60,"Moderate"; 0.61-0.80,