Rolling circle amplification RCA immunoassay

The manual RCA microarray immunoassay reported previously [4] was modified to optimize performance on an automated platform (Protedyne BioCube). Incubation times were increased from 30 to 45 min for two of the assay steps (RCA signal amplification and detector incubation), and the number and volume of washes between steps increased from 2 to 4-5 and from 20 to 30 mL, respectively. Slides were scanned using an LS200 scanner (TECAN). Scanned images were analyzed using proprietary software. The fluorescence intensity was analyzed for each sample analyte with the resulting mean intensity measurements converted into concentration values.

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