Streptavidin peroxidase

Concentrations of IgG and IgA in each saliva sample were quantified by ELISA using human IgA and IgG standards (10 to 100 μg.ml^-1; Invitrogen, Paisley, UK) to obtain a standard curve. Two dilutions (1:500 and 1:1000) of each saliva sample (50 μl) were prepared in PBS (0.1M, pH 7.0) (three technical replicates) and incubated for 18 h in 96 well flat-bottomed microtitre plate (Nunc MaxiSorp, Fisher Scientific, Loughborough, UK) at 4°C. Following incubation, wells were blocked with 200 μl of 1% bovine serum albumin in PBS for 30 min followed by three washes with PBST (PBS with Tween 20 at 0.1% v/v). Bound antibody was probed with 50 μl of 1:10,000 (in PBST) biotinylated (goat) anti-human IgG or IgA and incubated at RT for 45 min, followed by washing with PBST. Streptavidin-peroxidase (1:10,000 in PBST) (Roche, Hertfordshire, UK) was added to each well for 30 min and washed with PBST. O-phenyl diamine dihydrochloride (200 μl of 0.4 mg.ml^-1) in 0.05M phosphate-citrate buffer at pH 5.0, with hydrogen peroxide (40 μl, 20% (v/v)), was added to each well and incubated in the dark for 30 min before reading at 450 nm in a PowerWave XS plate reader (BioTek Instrumentation Inc., Bedfordshire, UK). All saliva samples were diluted before the positive selection procedure to give equal concentrations of IgA and IgG in each sample.

KC (Peprotech, 25 ng/reaction) was incubated with chamber fluid at 37°C for 14 h with or without Roche Protease Inhibitor as indicated. 2 x SDS PAGE sample buffer was added and samples were separated on 4–20% Tris-MOPS gels (Genscript), and transferred to PVDF membranes. KC was identified using a biotinylated rabbit anti-mouse KC antibody (Peprotech) followed by streptavidin peroxidase (Roche). Three samples from individual mice were analyzed in parallel.