Microarray layout and processing

Forty-eight identical antibody microarrays with up to 48 different capture antibodies were printed onto single glass microscope slides. Four such slides were mounted onto a slide holder effectively generating a microtiter plate with 384 spacing and an antibody microarray at the bottom of each well. On each slide, eight ofthe wells were incubated with standard mixtures of purified antigens in diluent, resulting in an eight-point standard titration curve that was used to quantify the analyte concentrations in each sample. The 40 remaining wells per slide were incubated with four dilutions (2-, 20-, 200-, and 200 000-fold) often samples. The diluent used throughout contained Roche "Complete" protease inhibitor cocktail at one tablet per 50 mL. After incubation for 1 h, all arrays were washed; a mixture of biotinylated detection antibodies was applied for 1 h, followed by washing. In a final 1 h incubation, RLS gold particles were applied to the arrays. Excess material was removed by washing. Slides were dipped twice into 50 mL deionized water and spun dry before coating with "RLS archiving" solution. For further details see Saviranta et al., [18].

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