The maturation of proteomics as a separate experimental field from genomics has highlighted the importance of obtaining information concerning a given protein’s activation state. This information can often be obtained by investigating the protein’s post-translational modification status—in particular, its phosphorylation state. The difficulties in producing high specificity and sensitivity affinity reagents specific for phospho-epitopes in sufficient quantities is, however, limiting the scope of diagnostic and research assays, where a multitude of phosphorylation states need to be probed simultaneously.
To address this reagent shortage, a series of high-throughput methods were devised at the EMBL Monoclonal Antibody Core Facility in Montorotondo, Italy. These methods incorporated robotic liquid handlers to perform somatic cell fusions at the rate of eight reactions per week—one or two is the norm—on lymphocytes derived from antigen-primed mouse spleens to generate classical hybridomas.
These eight fusions generated a total of 15,360 different samples to be tested by an affinity capture assay such as ELISA.
Successful screening for specificity against a phospho-epitope requires probing of both the phosphorylated and unphosphorylated form of the peptide, and a third, unrelated target, which has the same phosphorylated amino acid present in a different primary sequence context. This strategy ensures that only cell lines specific for the relevant amino acids are isolated, but triples the number of capture assays required to 46,080.
Furthermore, these assays must be completed within 48 hours to enable isolation of antibody-generating cell lines before the growth medium is exhausted. These combined issues push throughput requirements beyond the capacity of a standard plate-based ELISA, as the screen would necessitate the 480 96-well plates be processed.
First Step to the Solution
The most parallel assay technology to emerge in recent years is the microarray. The potential to perform a single test and yet obtain tens or hundreds of thousands of data points simultaneously, made microarrays an attractive solution to effectively manage throughput.
A novel antigen microarray assay was recently devised by EMBL in which a single peptide-coated glass microscope slide per test antigen (phosphorylated, unphosphorylated, and irrelevant peptide) would be printed with the cell culture supernatants from each fusion. At three slides per fusion, only 24 slides would be needed for eight fusions while maintaining the same level of screening complexity.
Initial screens using microarrays produced on a contact microarrayer proved problematic. Unreliable contact deposition necessitated triplicate printing of each supernatant; as the maximum spot density per slide was 17,280, only three fusions could be spotted onto each slide in a single print run (5,760 spots were required per fusion). This restriction limited print-run length and significantly decreased unattended operation time, while increasing manual intervention.