Miniaturization and automation of an ubiquitin ligase cascade enzyme-linked immunosorbent assay in 1,536-well format.

Enzyme-linked immunosorbent assays (ELISAs) are a long established and widely used assay format for drug discovery and diagnostics. They offer many advantages over homogeneous assay formats, including high sensitivity and separation (wash) steps that remove detection-interfering compounds. Many high-throughput screening assays are now performed in miniaturized formats (1,536- and 3,456-well plates) for higher throughput and lower reagent consumption. With miniaturization, separation steps in assays such as ELISA can become difficult to implement. Here we report on the implementation of the Kalypsys, Inc. (San Diego, CA) 1,536-well plate washer to enable the successful miniaturization and full automation of an ELISA that monitors ubiquitin ligase activity. The 1,536-well plate ELISA was robust and used for the high-throughput screening of a large screening collection (>1 million compounds).

A high-precision fluorogenic cholesteryl ester transfer protein assay compatible with animal serum and 3456-well assay technology.

Cholesteryl ester transfer protein (CETP) is a serum component responsible for both cholesteryl ester and triglyceride trafficking between high-density lipoprotein (HDL) and the apolipoprotein B (apoB)-containing very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL). Several fluorescence-based assays that monitor these transfers have been reported, but to date such assays have suffered from a low signal/background (S/B) ratio and have been described for use only in relatively purified in vitro systems. We have modified the more advanced of these assays to incorporate a noninterfering, nondiffusable fluorescence quencher into previously described cosonicate particles, often referred to as microemulsions. This simple improvement resulted in particles that had an average threefold enhanced S/B window over particles without quenchers but that continued to show the essential properties of a catalytic assay, including catalysis to a single endpoint, excellent linearity with protein and particle concentration, and an appropriate sensitivity to inhibition. This reduced assay noise allowed the subsequent development of protocols for the direct measure of cholesteryl ester (CE) transfer activity resident in human and animal serum as well as the development of 384- and 3456-well screening protocols with good precision and accuracy. Thus, by expanding the dynamic response window of the assay, we have created an assay generalizable to many settings.

Miniaturization of absorbance assays using the fluorescent properties of white microplates.

Miniaturization of high-throughput screening (HTS) assays has several obvious advantages, including increased throughput and lower cost by reduction in reagent consumption. Although absorbance assays are widely used in research laboratories, their application for HTS in a low-volume format has been met with mixed success because they are difficult to miniaturize. Challenges for the miniaturization of absorbance assays include low signal due to short path lengths and meniscus distortions in small well sizes. Here we describe a method to miniaturize absorbance assays to standard, white, low-volume 384-well and 1536-well microplates using a fluorometric plate reader for detection. The premise of this absorbance assay is based on the fluorescent properties of white microplates and the ability of a colored product to quench the fluorescence signal from the plate by absorbing either the excitation light or the emission light. This method was applied to the detection of inorganic phosphate using Quinaldine red and Malachite green dyes and to the monitoring of alkaline phosphatase hydrolysis of p-nitrophenyl phosphate. These assays can be carried out in low volumes, give robust screening statistics, and can be accomplished with a simple, inexpensive fluorometric plate reader.