Most critically, however, no user-friendly high-throughput screening tests had been developed for this class of enzymes. Current assay technology for targets similar to GOAT rely on radiolabeling techniques, which are labor-intensive and require additional safety precautions.

When Janda and Garner set out to develop an assay to monitor GOAT's activity, they aimed to change this situation.

Taking a Fresh Approach

The scientists were particularly interested in designing a fluorescence-based assay for GOAT. In contrast to radiolabeling techniques, fluorescence assays are safe, can easily be performed in a high-throughput format, and require only a simple light source.

In developing their new approach, the team was inspired by enzyme immunoassay strategies commonly used in biology, chemistry, and medicine, and by "click chemistry," a concept developed at Scripps Research. Because the amount of ghrelin modified by GOAT is minute, the scientists also used the principle of multi-turnover signal amplification for accurate detection.

In the end, the team succeeded in designing and creating the new technology-which they call "catalytic assay using enzyme-linked click chemistry assay" (cat-ELCCA).

"This new assay technology is both highly sensitive and reproducible," noted Janda, "making it an excellent assay for high-throughput screening."

Janda and Garner are now further developing the assay so that researchers can screen thousands to millions of compounds at once against the activity of GOAT.

Source: Scripps Research Institute