Modulation of nasopharyngeal innate defenses by viral coinfection predisposes individuals to experimental pneumococcal carriage

Glennie, S; Gritzfeld, J F; Pennington, S H; Garner-Jones, M; Coombes, N; Hopkins, M J; Vadesilho, C F; Miyaji, E N; Wang, D; Wright, A D; Collins, A M; Gordon, S B; Ferreira, D M

Levels of FH, lactoferrin, SLPI, and b-defensin 2 in nasal wash and anti-PspC in sera samples. To measure levels of FH, 96-well plates were coated with nasal wash samples serially diluted in carbonatebicarbonate buffer. Purified human FH (Calbiochem, Watford, UK) was used as a standard. Goat anti-FH antibodies (Calbiochem) followed by anti-goat IgG horseradish peroxidase-conjugated (R&D Systems, Abingdon, UK) were used for detection before development with TMB Substrate Reagent Set (BD, Oxford, UK). Levels of lactoferrin (AssayPro, St Charles, MO), SLPI (R&D), and b-defensin 2 (Antigenix America, Huntington Station, NY) were measured by sandwich ELISAs following the manufacturer's recommendation. Briefly, 96-well plates were coated with anti-human lactoferrin (AssayPro) or SLPI (R&D). Plates were blocked with phosphatebuffered saline (PBS)-1% bovine serum albumin before nasal wash samples serially diluted in PBS-0.1% bovine serum albumin were added. Human lactoferrin (AssayPro) and recombinant human SLPI (R&D) were used as standards. Biotinylated rabbit anti-lactoferrin or goat anti-SLPI followed by streptavidin-alkaline phosphatase (AbD Serotec, Kidlington, UK) were used for detection before development with 0.5 mg ml À 1 of P-nitrophenyl phosphate. Absorbance (450 nm) was measured for all assays using the FLUOstar OMEGA plate reader (BMG Labtech, Aylesbury, UK). The b-defensin 2 levels were measured using a kit as per the manufacturer's instructions (Antigenix America). Levels of anti-PspC IgG were measured by ELISAs using plates coated with recombinant purified PspC and a standard sera sample with known anti-PspC concentration as previously described using the last dilution of the sample with optical density 40.1 to calculate the sample concentration. 17 A four-parameter fit was used to generate the standard curve. All samples were run in triplicate, and samples with coefficient of variation of 415% were repeated.