Printing Low Density Protein Arrays in Microplates

Here, we provide methods for the creation of protein microarrays in microplates. The microplate consists of 96 wells with each well capable of holding a protein microarray at a spot density of up to 400 (20 � 20) individual elements. Arrays of capture monoclonal antibodies, corresponding to specific interleukins, were printed onto the bottom of the wells which had been surface activated for covalent attachment. A Biomek� 2000 laboratory automation workstation (Beckman Coulter, Inc., Fullerton, CA) equipped with a high-density replicating tool was used for printing low density 3 � 3 to 5 � 5 arrays. For higher density arrays, a microarrayer system (Cartesian PS7200, Genomic Solutions, Inc., Ann Arbor, MI) was employed. Multiple antigens were simultaneously analyzed without detectable cross-reactivity associated with capture antibody or secondary antibody interactions. Detection of interleukin antigens, spiked into cell culture media containing 10% fetal calf serum, was specific and sensitive.