Computational protocol: Recombinant polymorphic membrane protein D in combination with a novel, second-generation lipid adjuvant protects against intra-vaginal Chlamydia trachomatis infection in mice

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Protocol publication

[…] Serum was obtained on days 0, 35 and 56 following initial immunization and 6 weeks post challenge. 96-well Assay plates (Costar) were coated with 50 μl rPmpD (1 μg/ml) at 4 °C overnight in coating buffer (0.1 M Na2CO3, 0.1 M NaHCO3, pH 9.6). Plates were washed four times with 300 μl/well wash buffer (1× PBS, 0.05% Tween-20), and all subsequent washing steps carried out in identical fashion using an automated SkanWasher 400 (Molecular Devices, USA). Blocking buffer (1× PBS, 0.05% Tween-20, 0.1% BSA) was added (200 μl/well) and plates incubated for 2hr at room temperature prior to addition of serial dilutions of serum (100 μl/well) or cervico-vaginal lavage. After incubation for 2 h at 37 °C, plates were washed and then incubated with HRP-conjugated goat anti-mouse IgG, IgG1 or IgG2c antibodies (AbD Serotec) at 37 °C for 30 minutes. After the final wash step, 50 μl/well of 3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Sigma-Aldrich) was added. Colour development was stopped after 5 min with 1 M H2SO4 (KPL, USA) and plates were read immediately at 450 nm (OD450) with a VersaMax microplate reader in conjunction with the SoftMax Pro v.5.3 software (Molecular Devices, USA). Reciprocal serum dilutions corresponding to 50% maximal binding were used to obtain titres. [...] Differences between two independent means were determined with a two-tailed t-test using GraphPad Prism 6. For multiple comparisons, groups were compared using a one-way analysis of variance (ANOVA) following transformation of skewed distributions in STATA/IC 10 (StataCorp, USA). Sample size calculations were performed in G∗Power version 3.1 using both a priori analysis based on previous studies , and post hoc pilot experiments performed in our laboratory. A total of 5 animals were sufficient to detect expected effect sizes with a power of 0·8 (alpha = 0·05) for all experimental readouts. […]

Pipeline specifications

Software tools SoftMax Pro, G*Power
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Chlamydia Infections