Optimization of the surfaces used to capture antibodies from single hybridomas reduces the time required for microengraving.

The most common method for the generation of monoclonal antibodies involves the identification and isolation of hybridomas from polyclonal populations. The discovery of new antibodies for biochemical and immunohistochemical assays in a rapid and efficient manner, however, remains a challenge. Here, a series of experiments are described that realize significant improvements to an approach for screening large numbers of single cells to identify antigen-specific monoclonal antibodies in a high-throughput manner (10(5)-10(6) cells in less than 12 h). The soft lithographic process called microengraving yields microarrays of monoclonal antibodies that can be correlated to individual hybridomas; the cells can then be retrieved and expanded to establish new cell lines. The factors examined here included the glass slide used for the microarray, the buffer used to deposit capture antibodies onto the glass, the type of polyclonal antibodies used to capture the secreted antibodies, and the time required for microengraving. Compared to earlier reports of this method, these studies resulted in increased signal-to-noise ratios for individual elements in the microarrays produced, and a considerable decrease in the time required to produce one microarray from a set of cells (from 2-4 h to 3-10 min). These technical advances will improve the throughput and reduce the costs for this alternative to traditional screening by limiting serial dilution.